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E Handman

Publications and source records attributed to E Handman.

At least 73 records · Page 4Linked to original sources
PubMed1992

Changes in the precursor frequencies of IL-4 and IFN-gamma secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis.

Limiting dilution analysis was used to estimate the frequency of clonogenic Ag-specific CD4+ T lymphocytes in draining lymph nodes of mice over the course of infection with Leishmania major, and to measure the production of IL-2, IL-3, IL-4, IFN-gamma, and TNF by the resultant clones. Infection of both genetically susceptible BALB/c ("non-healer") and resistant C57BL/6 ("healer") mice resulted in at least a fourfold increase in the frequency (to about 0.3%) and at least a 10-fold increase in the total number of lymph node CD4+ cells that formed clones when cultured with L. major Ag in vitro. At 1 wk after infection, the majority of clones from BALB/c mice secreted IL-4 (precursor frequency 0.15%) and fewer secreted IFN-gamma (0.05%); this pattern remained constant for at least 8 wk after infection. In C57BL/6 mice, however, a high precursor frequency of IL-4-secreting clones was measured in the first 1 to 2 wk when the mice had lesions, but resolution of infection was associated with a decrease in the frequency of IL-4-secreting clones (from 0.13% at 2 wk to 0.03% at 4 wk) and an increase in the frequency of IFN-gamma-secreting clones (from 0.08% to 0.22%). At all stages of infection, most clones from either mouse strain secreted IL-3 and very few secreted TNF. Analysis of PCR-amplified cDNA from draining lymph nodes of infected mice also revealed that IL-4 and IFN-gamma mRNA were expressed in both mouse strains early in infection. IL-4 mRNA was the major species at 2 and 6 wk after infection in BALB/c mice, but declined relative to IFN-gamma mRNA over this time in C57BL/6 lymph nodes. Precursor frequency estimates of lymphokine-secreting CD4+ cells in draining lymph nodes therefore correlated with lymphokine expression patterns in vivo. Analysis of a panel of individual short term clones derived from mice 1 wk after infection revealed marked heterogeneity in lymphokine production patterns. In BALB/c mice, 49% secreted IL-4 without IFN-gamma, 18% secreted IFN-gamma without IL-4, and 14% secreted both IL-4 and IFN-gamma. Similarly in C57BL/6 mice, 39% secreted IL-4, 20% secreted IFN-gamma, and 17% secreted both lymphokines. Many of the clones also produced IL-3 and/or IL-2. Together the data suggest that both IL-4 and IFN-gamma are synthesized early in infection of susceptible and resistant mice as assessed by mRNA and precursor frequency analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes.

Leishmania are obligatory intracellular parasites in mammalian macrophages that gain entry by receptor-mediated phagocytosis. Their major cell surface glycoconjugate, lipophosphoglycan (LPG), has been implicated in this process. A monoclonal antibody specific for Leishmania major LPG (WIC 79.3), which has been shown to block promastigote attachment to macrophages, was used to identify a macrophage-binding determinant of LPG. WIC 79.3 bound exclusively to the phosphorylated repeats of LPG and not to the saccharide core or lipid anchor. Furthermore, the epitope recognized by WIC 79.3 mapped to the phosphorylated oligosaccharide P5b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-, which is unique to the LPG of promastigotes of L.major. Phosphorylated oligosaccharides P3, PO4-6[Gal(beta 1-3)[Gal(beta 1-4) Man(alpha 1-, and P4b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)] Gal(beta 1-4)Man(alpha 1-, were also recognized by WIC 79.3 but with considerably lower (approximately 100-fold) affinities. The phosphorylated oligosaccharide P5b inhibited attachment of promastigotes of L. major to the macrophage cell line J774 to the same degree as phosphoglycan (derived from LPG) and Fab fragments of WIC 79.3, suggesting that P5b is a site of L. major LPG that is recognized by macrophage receptor(s) and is an important determinant in the attachment of promastigotes to host macrophages and initiation of infection.

Animals↗
PubMed1992

Treatment of murine macrophages with interferon-gamma inhibits their ability to bind leishmania promastigotes.

The binding of leishmania promastigotes to macrophages pretreated with interferon-gamma (IFN-gamma) was compared to binding to untreated (resident) cells. IFN-gamma-treated macrophages bound fewer leishmania promastigotes than did untreated cells. The decreased binding was apparent over a wide dose range of parasite inocula when the assays were performed in the absence of exogenous complement. This decrease was specific to leishmania, since treated and untreated macrophages bound comparable amounts of immunoglobulin G- and complement-coated sheep red blood cells. Decreased parasite binding occurred early in the macrophage activation pathway. Pretreatment of macrophages with IFN-gamma for as little as 6 h, a time insufficient to induce other macrophage activation parameters, significantly reduced their ability to bind leishmania promastigotes. To determine the mechanism of this decreased phagocytosis by activated cells, macrophages were pretreated with specific inhibitors before the addition of leishmania. The binding of promastigotes to untreated (resident) macrophages was inhibited by approximately 50% by reagents that blocked either of two macrophage receptors, complement receptor type 3 (Mac-1) or a leishmania species-specific lectin-like receptor. Binding to IFN-gamma-treated macrophage populations, in contrast, was substantially inhibited only by antibody to Mac-1. Saccharides that were 50% inhibitory in the resident cell population, decreased binding by less than 10% in activated cells. The lack of saccharide inhibition by IFN-gamma-treated cells was also reflected in an inability of activated macrophages to bind to beads coated with purified leishmania lipophosphoglycan (LPG). These LPG-coated beads bound well to resident macrophages but poorly to activated cells. Thus, leishmania bind to macrophages by two distinct mechanisms, one that utilizes Mac-1 and a second mechanism that does not depend on complement and is saccharide inhibitable. These two binding mechanisms are distinct and differentially regulated in resident and activated cells.

Animals↗
PubMed1992

Cellular and humoral immune responses in a population from the Baringo District, Kenya to Leishmania promastigote lipophosphoglycan.

In a cross-sectional house-to-house study in a leishmaniasis-endemic area in Kenya, the cellular and humoral immune response to Leishmania lipophosphoglycan (LPG) was determined. Clinical data, peripheral blood mononuclear cells, and plasma were obtained from 50 individuals over the age of eight years. Lymphoproliferation and interferon-gamma (IFN-gamma) production by these cells were examined. It was shown that cells from all six individuals in the population with a history of kala-azar responded to LPG in the lymphocyte proliferation assay, and four of these six responded in the IFN-gamma assay. In contrast, cells from 12 of 44 individuals from the study area with no history of kala-azar and none of the five Danish control samples responded to LPG. Antibodies against LPG were detected by enzyme-linked immunosorbent assay in 45 of 50 plasma samples. Our findings clearly show that mononuclear cells from kala-azar patients cured of infection were able to respond to the LPG preparation. The finding of a specific cellular immune response to LPG in 12 of 44 individuals with no history of kala-azar is consistent with previous epidemiologic studies, in which it has been shown that a proportion of L. donovani infections run a subclinical course. The high frequency of individuals with antibodies against LPG might indicate that a majority of the population had been exposed to the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

An antigenically distinct lipophosphoglycan on amastigotes of Leishmania major.

We show that lipophosphoglycan (LPG) on the surface of amastigotes of Leishmania major is antigenically and biochemically distinct from promastigote LPG. A rabbit antiserum raised against the amastigote integral membrane fraction detected LPG spanning the region of Mr 55,000-100,000 on Western blots of the amastigote integral membrane fraction, but did not recognize the promastigote integral membrane fraction. WIC 79.3, a monoclonal antibody which recognizes L. major metacyclic promastigote LPG, did not recognize the amastigote integral membrane fraction on Western blots. The antigen recognized by this rabbit antiserum was shown to be LPG by its migration pattern on SDS-PAGE, the presence of terminal galactose residues, recognition by a monoclonal antibody to LPG, WIC 108.3, the biosynthetic incorporation of label from [3H]glucose and [32P]phosphate, a hydrophobic chromatography elution profile similar to promastigote LPG, and the presence of a lipid anchor sensitive to phosphatidylinositol-specific phospholipase C. The temporal regulation of LPG expression during parasite differentiation was studied in vitro. During amastigote-to-promastigote transformation, the amastigote-specific form of LPG disappeared after subculture at 48 h. The WIC 79.3 epitope was not detected by Western blotting on transforming parasites until 48 h in culture. During promastigote-to-amastigote transformation, the amastigote-specific form of LPG was detected 12 h after infection. WIC 79.3 epitopes gradually diminished over 48 h. The results demonstrate the developmentally regulated expression of an antigenically distinct LPG on amastigotes of L. major.

Animals↗
PubMed1991

Structure and antigenicity of the lipophosphoglycan from Leishmania major amastigotes.

The lipophosphoglycan (LPG) of the intracellular amastigote form of the protozoan parasite Leishmania major is chemically distinct from the LPG on the surface of the extracellular promastigote form. Amastigote LPG is composed of the monosaccharides galactose, glucose, mannose, glucosamine and inositol in the molar ratio 51:30:24:1:1; arabinose is absent. The lipid anchor comprises four alkylglycerols, with alkyl chain lengths 24:0, 22:0, 20:0 and 26:0 in the molar ratio 68:18:8:6. Phosphate is present at 4% w/w of total carbohydrate. HPLC gel permeation reveals LPG to be a polydisperse family of molecules Mr 100-6 kDa. The results from immunological studies with LPG-directed antibodies are consistent with amastigote LPG having the expected tripartite structure of GPI-anchor, a core glycan and the phosphorylated disaccharide repeat backbone. Human sera from L. major patients bound amastigote LPG in enzyme-linked immunosorbent assays.

Animals↗
PubMed1991

Activation of human T lymphocytes by Leishmania lipophosphoglycan.

This study describes Leishmania antigen-induced activation of lymphocytes isolated from Kenyan donors, previously treated for visceral leishmaniasis, and from Danish and Kenyan controls. Peripheral blood mononuclear cells (PBMC) from cured Kala-Azar patients proliferated and produced Interferon-gamma in vitro in response to lipophosphoglycan (LPG) isolated from Leishmania major. The proliferative response was mainly due to activation of CD2-positive T cells. PBMC from controls did not respond to LPG, but to sonicates prepared from both L. major and L. donovani promastigotes. The surface glycoprotein GP 63 failed to activate PBMC from any of the donors tested. These results show that the individuals cured from visceral leishmaniasis had expanded T-cell clones recognizing LPG, conceivably as a result of Leishmania infection. The LPG preparation was without detectable protein contamination. Thus, the results suggest that human T lymphocytes can respond to glycolipid antigens.

Animals↗
PubMed1990

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains.

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.

Animals↗
PubMed1990

Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice.

The Mr 63,000 membrane polypeptide (gp63) is one of the Leishmania receptors for host macrophages and has been shown to protect mice from infection. The gene encoding gp63, the major Mr 63,000 surface glycoprotein of L. major promastigotes, has been expressed as a fusion protein with the enzyme glutathione S- transferase encoded by the parasitic helminth Schistosoma japonicum. This fusion protein was recognized by polyclonal antibodies to the native Leishmania gp63 polypeptide. The insoluble gp63 fusion protein was purified by SDS-PAGE and electroelution and was used to raise antibodies in rabbits. These rabbit anti-gp63 antibodies recognized the fusion protein and the denatured parasite gp63 on immunoblots and by immunofluorescence on fixed promastigotes, but did not recognize the native molecule on live organisms. However, antibodies raised against native promastigote glycoproteins, affinity purified on solid-phase gp63 fusion protein, recognized both native and denatured gp63, suggesting the presence of native determinants in the recombinant protein. The gp63 fusion protein did not protect mice of either healer or nonhealer phenotype from challenge infection with live promatigotes. The implications of these results for the engineering of recombinant DNA-produced molecular vaccines are discussed.

Animals↗
PubMed1990

Lipophosphoglycan expression and virulence in ricin-resistant variants of Leishmania major.

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats jointed by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the Mr 63,000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies. All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.

Animals↗
PubMed1990

Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies.

Leishmania major infection of macrophages is followed by a time-dependent appearance of lipophosphoglycan (LPG) that can be detected on the surface of infected cells by monoclonal antibodies. The origin of these LPG epitopes is probably the intracellular amastigote. LPG epitopes could be detected on the amastigote and the infected macrophage by a number of monoclonal antibodies directed to several distinct determinants on the phosphoglycan moiety. The macrophage-expressed LPG may be modified because, unlike the parasite LPG as expressed on promastigotes or amastigotes, it could not be radiolabeled by galactose oxidase or periodate treatment of infected cells followed by reduction with 3H-labeled sodium borohydride. Some LPG epitopes displayed on the macrophage may be anchored with glycosylphosphatidylinositol, and some may be in the water-soluble phosphoglycan form bound to macrophage integrins involved in its specific recognition. The water-soluble population could be released from the infected macrophage by gentle protease treatment.

Animals↗
PubMed1989

The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen.

Polyclonal rabbit antiserum to the Triton X-114 phase material of Leishmania major, which comprises the surface and internal integral membrane proteins of the parasite, was used to screen a lambda gt11 genomic expression library. A recombinant clone producing a Mr 123,000 beta-galactosidase fusion protein was isolated. Antibodies affinity-purified on this fusion protein recognized a complex of three surface-oriented proteins of promastigotes of L. major of Mr 94,000, 90,000, and 80,000 that we have termed the promastigote surface Ag 2 (PSA-2) complex. The DNA sequence of the insert in this clone predicted the 3' end of an open reading frame encoding a hydrophobic C-terminus. The inferred C-terminal sequence was suggestive of a glycosylphosphatidyl-inositol membrane anchoring mechanism. Phosphatidylinositol-specific phospholipase C treatment of the native PSA-2 proteins caused a shift in their electrophoretic mobility with an apparent reduction in the molecular weight of the PSA-2 complex. After phospholipase C treatment these proteins also displayed the cryptic cross-reacting determinant recognized by antibodies to the Trypanosoma brucei variant surface Ag. Moreover, PSA-2, which previously partitioned in the detergent phase after Triton X-114 phase separation, became water-soluble after phospholipase C treatment. Immunoprecipitation of the PSA-2 proteins with sera directed to lectin-binding proteins indicated that these polypeptides may be differentially glycosylated. Finally, these PSA-2 proteins were recognized by sera from some patients with cutaneous leishmaniasis.

Amino Acid Sequence↗
PubMed1989

Evidence of T-cell recognition in mice of a purified lipophosphoglycan from Leishmania major.

We have previously reported that a Leishmania major lipophosphoglycan (LPG), given with killed Corynebacterium parvum as an adjuvant, can vaccinate mice against cutaneous leishmaniasis. In order to analyze whether T cells are able to recognize this important parasite antigen, we have studied both humoral and cellular immune responses to L. major LPG that had been isolated from promastigotes by sequential solvent extraction and hydrophobic chromatography. The data show that immunization of mice with highly purified LPG induced an increase in frequency of L. major-reactive T cells and the production of immunoglobulin G antibodies to LPG. Furthermore, genetically resistant mice infected with L. major were able to develop a specific delayed-type hypersensitivity response in the ear to L. major LPG. These findings strongly suggest that T cells can recognize and respond to glycolipid antigens, in this case a host-protective Leishmania LPG, even though such antigens appear not to be potent T-cell stimulators in mice.

Animals↗
PubMed1989

Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens.

Animals↗
PubMed1988

Immunochemical characterization of a glyco-inositol-phospholipid membrane antigen of Leishmania major.

A low m.w. polymorphic glyco-inositol-phospholipid (GIPL) of Leishmania major was studied by using three different mAb. This molecule is shown to be distinct from the previously described lipophosphoglycan of L. major in its m.w., antigenic properties, expression during parasite growth, and kinetics of synthesis and catabolism. GIPL is shown to be released from the parasite surface in a water-soluble form, probably by an endogenous phospholipase. GIPL is also detectable on the surface of infected macrophages, although not all epitopes are detectable in this state. GIPL can be metabolically labeled with [3H]galactose, [3H]inositol, [32P]phosphate, and [3H]palmitic acid. GIPL can also be labeled on the surface of living promastigotes with galactose oxidase and [3H]sodium borohydride. The kinetics of synthesis and catabolism are much faster than those of lipophosphoglycan. GIPL is sensitive to degradation upon parasite lysis and becomes undetectable by mAb after 20 h at 37 degrees C. The expression of GIPL on the surface of promastigotes is more abundant during the logarithmic phase of growth, and declines in stationary phase.

Animals↗
PubMed1988

GM-CSF produced by recombinant vaccinia virus or in GM-CSF transgenic mice has no effect in vivo on murine cutaneous leishmaniasis.

The hemopoietic growth and differentiation regulators, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the multipotential stimulating factor (multi-CSF) have been shown to have major effects on the effector function of mature macrophages. In this study we have examined the effect of recombinant GM-CSF and multi-CSF expressed transiently from recombinant vaccinia virus, or constitutively in GM-CSF transgenic mice on the development of cutaneous leishmaniasis, caused by Leishmania major in genetically susceptible or resistant mice. We observed no effect on the development of lesions when GM-CSF or multi-CSF were administered before infection, nor on the healing of lesions when they were administered after appearance of lesions. Although only some of the GM-CSF transgenic mice or their normal littermates developed lesions after infection with L. major, there was no difference between the groups in the rate of lesion development or in the size of lesions.

Animals↗
PubMed1987

Heterologous protection in murine cutaneous leishmaniasis.

Mice immunized with a glycolipid antigen (GL) of Leishmania major plus adjuvant are relatively resistant to subsequent infection with this protozoan parasite. The GL is affinity purified on the monoclonal antibody WIC-79.3 which is L. major-specific and does not react with L. donovani. When another monoclonal, WIC-108.3, which cross-reacts with several Leishmania species, is used to affinity purify GL from L. donovani, the eluted material can partially protect genetically resistant mice against L. major. Thus, GL cross-reactions may in part underlie the known protective effects of crude L. donovani antigens against L. major infection. Experiments with live parasites of the L. major isolate LRC-L119, that is non-pathogenic in mice, that does not survive long in macrophages in vitro, and that has not been shown to contain any WIC-79.3 reactive GL, indicated that this isolate will very effectively protect mice against subsequent infection. This raises the possibility that GL is only one of at least two different classes of vaccinating antigen capable of protectively immunizing mice in this cutaneous leishmaniasis model.

Animals↗