Differential recognition of Leishmania aethiopica antigens by lymphocytes from patients with local and diffuse cutaneous leishmaniasis. Evidence for antigen-induced immune suppression

Data are presented to suggest that differential Ag expression by parasites derived from diffuse (DCL) vs local (LCL) cutaneous leishmaniasis patients may be responsible for the Ag-specific anergy seen in DCL patients. The evidence suggests that promastigotes derived from DCL patients express epitopes which preferentially stimulate suppressor activities in DCL patients. These determinants appear to be expressed less, if at all by promastigotes derived from LCL patients. The Ag-specific suppression or nonresponsiveness which dominates the immune response in DCL patients during an active infection can be abrogated by drug treatment or removal of live DCL parasites, which suggests that Ag-induced regulatory cells, probably of T cell lineage, are most likely responsible for the nonresponsiveness seen in untreated DCL patients. Thus the mechanisms of immune regulation operating in this disease differ from that of lepromatous leprosy where the specific unresponsiveness (anergy) is irreversible even after successful treatment.     

Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) presents distinct active clinical forms with different grades of severity, known as localised (LCL), intermediate (ICL) and diffuse (DCL) cutaneous leishmaniasis. LCL and DCL are associated with a polarised T-helper (Th)1 and Th2 immune response, respectively, whereas ICL, or chronic cutaneous leishmaniasis, is associated with an exacerbated immune response and a mixed cytokine expression profile. Chemokines and chemokine receptors are involved in cellular migration and are critical in the inflammatory response. Therefore, we evaluated the expression of the chemokines CXCL10, CCL4, CCL8, CCL11 and CXCL8 and the chemokine receptors CCR3, CXCR3, CCR5 and CCR7 in the lesions of patients with different clinical forms of ACL using immunohistochemistry. LCL patients exhibited a high density of CXCL10+, CCL4+ and CCL8+ cells, indicating an important role for these chemokines in the local Th1 immune response and the migration of CXCR3+ cells. LCL patients showed a higher density of CCR7+ cells than ICL or DCL patients, suggesting major dendritic cell (DC) migration to lymph nodes. Furthermore, DCL was associated with low expression levels of Th1-associated chemokines and CCL11+ epidermal DCs, which contribute to the recruitment of CCR3+ cells. Our findings also suggest an important role for epidermal cells in the induction of skin immune responses through the production of chemokines, such as CXCL10, by keratinocytes.     

Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani(1)

To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family was identified and characterised. The GRP78 gene was localised to chromosome 15 in L. donovani, L. major, and L. mexicana by pulse-field gel electrophoresis. The Leishmania GRP78 protein contain a carboxy-terminal endoplasmic reticulum retention signal sequence (MDDL) as does the Trypanosoma cruzi GRP78. Immunofluorescence using antibodies to the recombinant DNA-derived GRP78 protein showed staining localised to reticular material throughout the cytoplasm and in the perinuclear region of promastigotes, suggesting that the protein is localised in the endoplasmic reticulum. The protective efficacy of GRP78 was assessed in mice vaccine experiments. A GRP78 DNA vaccine primed for an immune response that protected C57Bl/6 and C3H/He mice against infection with L. major. Similarly vaccination with a recombinant form of GRP78 purified from Escherichia coli and administered with Freund's as adjuvant induced protective immunity in C57Bl/6 mice.     

Immunotherapy as a treatment of American cutaneous leishmaniasis: preliminary studies in Brazil.

A prophylactic vaccine composed of killed promastigotes of five stocks of Leishmania was tested as an immunotherapeutic agent against American cutaneous leishmaniasis (ACL). The agent was administered by deep intramuscular injection daily for 10 days, followed by a 10-day interval. Out of 62 patients so treated, 47 (76%) were considered clinically cured; 41 required 2-10 treatment courses and the other six 11-19 courses. None of the patients treated by immunotherapy displayed adverse side-effects. Immunotherapy proved to be effective in the treatment of single cutaneous lesions, multiple cutaneous lesions and in cases of mucocutaneous leishmaniasis. In comparison with chemotherapy (Glucantime), immunotherapy is less efficient and more prolonged but can be safely used when antimonials are contra-indicated or are found to be ineffective. Consideration is given to the treatment of victims of ACL living in rural areas remote from a medical centre.     

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.     

Exposure of phosphatidylserine on Leishmania amazonensis isolates is associated with diffuse cutaneous leishmaniasis and parasite infectivity

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of leishmaniasis, characterized by an inefficient parasite-specific cellular response and heavily parasitized macrophages. In Brazil, Leishmania (Leishmania) amazonensis is the main species involved in DCL cases. In the experimental model, recognition of phosphatidylserine (PS) molecules exposed on the surface of amastigotes forms of L. amazonensis inhibits the inflammatory response of infected macrophages as a strategy to evade the host immune surveillance. In this study, we examined whether PS exposure on L. amazonensis isolates from DCL patients operated as a parasite pathogenic factor and as a putative suppression mechanism of immune response during the infection. Peritoneal macrophages from F1 mice (BALB/c×C57BL/6) were infected with different L. amazonensis isolates from patients with localized cutaneous leishmaniasis (LCL) or DCL. DCL isolates showed higher PS exposure than their counterparts from LCL patients. In addition, PS exposure was positively correlated with clinical parameters of the human infection (number of lesions and time of disease) and with characteristics of the experimental infection (macrophage infection and anti-inflammatory cytokine induction). Furthermore, parasites isolated from DCL patients displayed an increased area in parasitophorous vacuoles (PV) when compared to those isolated from LCL patients. Thus, this study shows for the first time that a parasite factor (exposed PS) might be associated with parasite survival/persistence in macrophages and lesion exacerbation during the course of DCL, providing new insights regarding pathogenic mechanism in this rare chronic disease.     

Lipid and Lipopolysaccharide-Like Antigens of Leishmania Promastigotes1

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: 1) lipids, where the activity was determined by radioimmunoassay; 2) a lipopolysaccharide-like (LPS-like), water-soluble precipitate, where activity was determined both by radioimmunoassay and double gel diffusion, and 3) 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 (Kinetoplastida): species typing with isoenzyme and PCR-RFLP from cutaneous leishmaniasis patients in Ethiopia

Cutaneous leishmaniasis (CL) is an increasing public health problem in Ethiopia. There is a concern that it is spreading with increased incidence. In this study, we used isoenzyme electrophoresis and internal transcribed spacer one (ITS1) PCR-RFLP techniques to identify Leishmania species from CL patients in Ethiopia. We obtained isolates from 55 localized cutaneous leishmaniasis (LCL), 3 diffused cutaneous leishmaniasis (DCL) and 36 biopsy samples from 34 LCL and 2 DCL cases from All Africa Leprosy and Tuberculosis Rehabilitation and Training Center (ALERT) and clinically diagnosed CL cases from Ochollo village. Both isoenzyme and ITS1 PCR-RFLP techniques showed that Leishmania aethiopica (L. aethiopica) was the aetiologic agent in all cases. Our study also showed that ITS1 PCR-RFLP could identify Leishmania species from biopsy samples and suggests the method could be used for epidemiological surveillance of leishmaniasis in Ethiopia and for species-specific diagnosis.     

A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.     

Immunologic responsiveness in American cutaneous leishmaniasis lesions

American cutaneous leishmaniasis is a disease of skin and mucous membranes in which T lymphocytes reactive to Leishmania (Viannia) braziliensis are thought to contribute to protective immunity. To characterize the nature of the T cell inflammatory infiltrate in American cutaneous leishmaniasis lesions, immunohistochemistry with mAb that define T cell subpopulations and in situ hybridization to detect mRNA coding for IFN-gamma were performed. In both localized cutaneous (LCL) and mucocutaneous (MCL) lesions, we observed a predominance of T memory (CD4+CD45RO+) as compared to T naive cells (CD+CD45RA+). The percentages of cells containing IFN-gamma mRNA were equivalent in both LCL and MCL lesions. T cells were extracted from LCL and MCL lesions and analysis indicated that T cells from both lesions had been stimulated by L. (V.) braziliensis in vivo and gave equivalent proliferative responses in vitro. The present data suggest that T memory cells, which are likely to elaborate IFN-gamma, are components of DTH response to L. (V.) braziliensis and participate in the pathogenesis of both LCL and MCL lesions.     

NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

Leishmania mexicana causes localized (LCL) or diffuse cutaneous leishmaniasis (DCL). The cause of dissemination in DCL remains unknown, yet NK cells possibly play a role in activating leishmanicidal mechanisms during innate and adaptive immune responses. We had previously shown that Leishmania lipophosphoglycan (LPG) is a ligand for TLR2, activating human NK cells. We have now analyzed NK cells in LCL and DCL patients. NK numbers and effector mechanisms differed drastically between both groups of patients: DCL patients showed reduced NK cell numbers; diminished IFN-γ and TNF-α production; and lower TLR2, TLR1, and TLR6 expression as compared to LCL patients. The altered protein expression found in NK cells of DCL patients correlated with their down-regulation of IFN-γ gene expression in LPG-stimulated and non-stimulated cells as compared to LCL patients. NK cell response was further analyzed according to gender, age, and disease evolution in LCL patients showing that female patients produced higher IFN-γ levels throughout the disease progression, whereas TLR2 expression diminished in both genders with prolonged disease evolution and age. We furthermore show the activation pathway of LPG binding to TLR2 and demonstrated that TLR2 forms immunocomplexes with TLR1 and TLR6. In addition to the reduced NK cell numbers in peripheral blood, DCL patients also showed reduced NK cell numbers in the lesions. They were randomly scattered within the lesions, showing diminished cytokine production, which contrasts with those of LCL lesions, where NK cells produced IFN-γ and TNF-α and were found within organized granulomas. We conclude that in DCL patients the reduced NK-cell numbers and their diminished activity, evidenced by low TLR expression and low cytokine production, are possibly involved in the severity of the disease. Our results provide new information on the contribution of NK cells in Leishmania infections of the human host.     

Down-Regulation of TLR and JAK/STAT Pathway Genes Is Associated with Diffuse Cutaneous Leishmaniasis: A Gene Expression Analysis in NK Cells from Patients Infected with Leishmania mexicana

An important NK-cell inhibition with reduced TNF-α, IFN-γ and TLR2 expression had previously been identified in patients with diffuse cutaneous leishmaniasis (DCL) infected with Leishmania mexicana. In an attempt to pinpoint alterations in the signaling pathways responsible for the NK-cell dysfunction in patients with DCL, this study aimed at identifying differences in the NK-cell response towards Leishmania mexicana lipophosphoglycan (LPG) between patients with localized and diffuse cutaneous leishmaniasis through gene expression profiling. Our results indicate that important genes involved in the innate immune response to Leishmania are down-regulated in NK cells from DCL patients, particularly TLR and JAK/STAT signaling pathways. This down-regulation showed to be independent of LPG stimulation. The study sheds new light for understanding the mechanisms that undermine the correct effector functions of NK cells in patients with diffuse cutaneous leishmaniasis contributing to a better understanding of the pathobiology of leishmaniasis.     

CD8 cells of patients with diffuse cutaneous leishmaniasis display functional exhaustion: the latter is reversed, in vitro, by TLR2 agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular "exhaustion" described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.     

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease.

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the disease. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9, and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative mobility (Mr) or the quantitative amount (intensity) of the antigenic determinants. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patients' sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and the clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania braziliensis: effects of bacteria (Staphylococcus aureus and Pasteurella multocida) on the developing cutaneous leishmaniasis lesion in the golden hamster

Experimentally induced lesions of cutaneous leishmaniasis and the effect of concurrent bacterial infection on the development of these lesions were studied in the golden hamster. Male outbred golden hamsters received intradermal injections at the base of the tail with approximately 10(7) promastigotes of Leishmania braziliensis panamensis, or promastigotes combined with Staphylococcus aureus or Pasteurella multocida or both, bacteria only, or sterile Eagle's minimal essential medium (MEME). The size of the resulting lesions was measured at least twice each week. Hamsters were killed at postinoculation Days 6, 13, 20, 27, 41, or 48, and each lesion was measured, aseptically excised, and bisected; half was used for bacteriologic culture and the other half was prepared for light microscopic examination. Lesions resulting from L. b. panamensis alone progressed from initial erythema to a granulomatous nodule and finally to a necrotic granuloma, often capped by a crateriform ulcer. Lesions resulting from a suspension of L. b. panamensis with added S. aureus or S. aureus and P. multocida, were initially larger, more erythemic and contained a greater proportion of neutrophils up to postinoculation Days 14-21 than did lesions resulting from L. b. panamensis alone. Concurrent infections with bacteria such as S. aureus and P. multocida had little effect on the development of ulcerating characteristics of lesions, but when S. aureus was present it appeared to enhance the severity of the early lesions. Between postinoculation Days 14-28, lesions produced by L. b. panamensis, with or without added bacteria had similar developmental progression of sufficient size for optimal testing of antileishmanial compounds.     

The influence of natural rubber/Au nanoparticle membranes on the physiology of Leishmania brasiliensis

The development of nanotechnology has generated new means of disease diagnosis and treatment. Infectious diseases, including leishmaniasis, malaria, etc., have benefited from the advent of new nanomaterials and/or nanodevices capable of detecting specific antigens and antibodies with high specificity and low cost. In this paper, we present an investigation on a single-celled protozoan Leishmaniasis parasite, a disease considered of standard infectivity, given the high degree of immunological specificity. Natural rubber (NR) membranes incorporating gold nanoparticles (GNPs) were placed in the culture medium and the physiological behavior of Leishmania brasiliensis promastigotes was evaluated. The natural rubber membranes containing GNPs decreased the population growth rate, showing a lower index of living promastigotes (attached to the membrane surface) depending on the amount of nanoparticles deposited in the membrane surface. Such membranes may be used to develop a flexible band-aid for skin lesions from degenerative infection state, inhibiting the population growth of parasites in the lesions. In addition, natural rubber membranes would also stimulate angiogenesis in damaged tissues.     

Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major

Mitchell G. F., Handman E. and Spithill T. W. 1985. Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major. International Journal for Parasitology15: 677–684. In vaccination experiments not involving adjuvants, genetically-susceptible mice were injected with living avirulent cloned promastigotes of Leishmania major or killed promastigotes prior to cutaneous challenge with virulent cloned promastigotes. Emphasis was placed on aspects that may contribute to marked variability between experiments and between laboratories in vaccination of mice against cutaneous leishmaniasis. One variable, the challenge promastigotes, was shown to be important in that cloned virulent parasites (V121) were less pathogenic in terms of rate of cutaneous lesion development, than the parental isolate LRC-L137 when low doses of promastigotes were used and particularly when harvested from the log phase of culture. It is likely that avirulent parasites in mixed isolates can increase the rate of lesion development after cutaneous deposition. As reported previously, intraperitoneal, and more particularly intravenous injections of living avirulent cloned parasites (A12) increase resistance in mice. Most importantly, a difference has been demonstrated in the vaccinating efficacy of killed promastigotes of various isolates injected intravenously. This implies that certain isolates of L. major (e.g. the “Moshkovsky strain”) express “host-protective antigens” at higher levels, or in a qualitatively different manner, than other isolates (e.g. LRC-L137). The finding will greatly facilitate the identification of vaccine antigens in this system using immunochemical and gene cloning approaches.     

Leishmania amazonensis infection in nude mice.

Leishmania amazonensis is an intracellular protozoan parasite of macrophages. Cutaneous leishmaniasis in an immunocompetent host begins as papules or nodules followed by ulceration at the site of promastigote inoculation. In this study, the pathological changes of cutaneous leishmaniasis lesions in T cell deficient nude mice were examined. When infected with L. amazonensis promastigotes, nude mice developed non-ulcerative cutaneous nodules. By histological examination of cutaneous lesions, massive accumulation of vacuolated histiocytes containing amastigotes was observed in all the nude mice. Although infiltration of mononuclear and polymorphonuclear cells was seen in the lesions of immunocompetent mice, few such cells were observed in the lesions of nude mice. These results indicate the importance of T cells on the ulcer formation in cutaneous leishmaniasis.     

Virulence attenuation of a UDP-galactose/<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β1,4 galactosyltransferase expressing <Emphasis Type="Italic">Leishmania donovani</Emphasis> promastigote

Protozoan parasites of the genus Leishmania are the causative agent of leishmaniasis, a disease whose manifestations in humans range from mild cutaneous lesions to fatal visceral infections. Human visceral leishmaniasis is caused by Leishmania donovani. Long-term culture in vitro leads to the attenuation of the parasite. This loss of parasite virulence is associated with the expression of a developmentally regulated UDP-Galactose/N-acetylglucosamine beta 1-4 galactosyltransferase and galactose terminal glycoconjugates as determined by their agglutination with the pea nut agglutinin (PNA). Thus, all promastigotes passaged for more than 11 times were 100% agglutinated with PNA, and represent a homogeneous population of avirulent parasites. Identical concentrations of PNA failed to agglutinate promastigotes passaged for < or =5 times. These PNA(-) promastigotes were virulent. Promastigotes passaged from 5 to 10 times showed a mixed population. The identity of populations defined by virulence and PNA agglutination was confirmed by isolating PNA(+) avirulent and PNA(-) virulent clones from the 7th passage promastigotes. Only the PNA(+) clones triggered macrophage microbicidal activity. The PNA(+) clones lacked lipophosphoglycan. Intravenous administration of [(14)C] galactose-labeled parasite in BALB/c mice resulted in rapid clearance of the parasite from blood with a concomitant accumulation in the liver. By enzymatic assay and RT-PCR we have shown the association of a UDP-Galactose/N-acetylglucosamine beta1,4 galactosyltransferase with only the attenuated clones. By immunofluorescence we demonstrated that the enzyme is located in the Golgi apparatus. By western blot analysis and SDS-PAGE of the affinity-purified protein, we have been able to identify a 29 KDa galactose terminal protein from the avirulent clones.