Dynamic flux of microvesicles modulate parasite-host cell interaction of Trypanosoma cruzi in eukaryotic cells.

Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell-derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP-1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell-derived trypomastigote (tissue culture-derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP-1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP-1-derived MVs can fuse with parasite-derived MVs. Furthermore, MVs derived from the TCT-THP-1 interaction showed a higher fusogenic capacity than those from META- or EPI-THP-1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite-THP-1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP-1-derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology.

Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms.

SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMO(T106K) version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.

Are grape juices more erosive than orange juices?

AIMS: To evaluate the chemical characteristics of grape and orange juices, and their erosive potential in the decrease of microhardness and the loss of enamel structure. METHODS: Five grape and orange juices were evaluated for pH, titratable acidity, calcium, phosphate, and fluoride concentration. De-ionised water and Cola soft drink were used as a negative and positive control, respectively. Twelve specimens of bovine enamel were immersed in beverages for 10 min at 37 °C, 3 times/day for 7 days. Erosive potential was quantified using microhardness and loss of enamel structure. Anova One Way, Student's t test, Multiple Regression and Spearman Correlation (p < 0.05) were used to analyse the results. RESULTS: Powdered grape juice showed the lowest pH (3.18 ± 0.03) and pure grape juice presented the highest titratable acidity (5.48 ± 0.06 mL NaOH/100 mL). Fresh orange juice and soya-based grape juice revealed the lowest calcium (0.77 ± 0.12 mmol/L) and phosphate concentrations (0.35 ± 0.06 mmol/L), respectively. Among juices, powdered orange juice caused the greatest decrease in surface microhardness (SMH) (127.99 ± 40.47 ΔSMH) and grape juice from concentrate caused the greatest loss of enamel structure (13.30 ± 3.56 µm). CONCLUSIONS: All of the evaluated juices contributed to dental erosion. Grape juices presented greater erosive potential than orange juices. Pure, powdered and concentrated grape juices showed similar loss of enamel structure to the Cola soft drink. The erosive potential of beverages was statistically correlated to pH, titratable acidity, calcium, phosphate and fluoride concentrations.

Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses.

A strong activation of macrophages is observed during acute infection with Trypanosoma cruzi. Little is known, however, about the parasite molecules that are responsible for this early activation of innate immunity. Recent studies have shown the stimulatory activity of protozoan-derived glycosylphosphatidylinositol (GPI) anchors on cultured macrophages. In this review, we provide a detailed analysis of the correlation between structure and proinflammatory activity by T. cruzi-derived GPI anchors. We also cover the studies that have identified the Toll-like receptor 2 as a functional GPI receptor and have partially characterized signaling pathways triggered by T. cruzi-derived GPI anchors, which lead to the synthesis of proinflammatory cytokines in macrophages. Finally, we discuss the implications of these findings in resistance and pathogenesis during the infection with T. cruzi.

Decrease in sulphated glycosaminoglycans in aortic dissection--possible role in the pathogenesis.

STUDY OBJECTIVE: The aim was to investigate alterations in sulphated glycosaminoglycans in aortic dissection. DESIGN: Aortic fragments were taken from 10 patients within the first 3 d after onset of symptoms of aortic dissection and from nine age matched patients with no aortic disease. Sulphated glycosaminoglycans were analysed and quantified by agarose gel electrophoresis and densitometry after degradation with specific enzymes. MEASUREMENTS AND MAIN RESULTS: The amount of chondroitin sulphate was similar (7.14 v 7.60 mg.g-1 of dry tissue, n = 10, p greater than 0.5) in patients with dissection and in the control group. Total sulphated glycosaminoglycan content was decreased (11.51 v 14.26 mg.g-1 of dry tissue, n = 10, p less than 0.001). This difference was due to heparan sulphate (1.79 v 2.48 mg.g-1 of dry tissue, n = 10, p less than 0.05) and mainly to dermatan sulphate (2.58 v 4.18 mg.g-1 of dry tissue, n = 10, p less than 0.001). The ratio of 6-/4-sulphated disaccharides after chondroitinase ABC digestion was increased in the affected group. No correlation between these biochemical results and a histological evaluation of mucoid content was found. On the other hand, a significant increase in chondroitin sulphate could be observed related to aging. CONCLUSIONS: The diminution in sulphated glycosaminoglycans and its possible relationship with fat, collagen, and other extracellular matrix molecules could lead to a weakness in the aortic wall related to the dissection.

CO(2) and N(2)O emissions in a soil chronosequence at a glacier retreat zone in Maritime Antarctica.

Studies of C cycle alterations are extremely important to identify changes due to climate change, especially in the polar ecosystem. The objectives of this study were to (i) examine patterns of soil CO2-C and N2O-N emissions, and (ii) evaluate the quantity and quality of soil organic matter across a glacier retreat chronosequence in the Maritime Antarctica. Field measurements were carried out during January and February 2010 (summer season) along a retreating zone of the White Eagle Glacier, at King George Island, Maritime Antarctica. Soil samples (0-10cm) were collected along a 500-m transect at regular intervals to determine changes in soil organic matter. Field CO2-C emission measurements and soil temperature were carried out at regular intervals. In addition, greenhouse gas production potentials were assessed through 100days laboratory incubations. Soils exposed for a longer time tended to have greater concentrations of soluble salts and possess sandier textures. Total organic C (3.59gkg(-1)), total N (2.31gkg(-1)) and labile C (1.83gkg(-1)) tended to be lower near the glacier front compared with sites away from it, which is correlated with decreasing degree of humification of the soil organic matter with exposure time. Soil CO2-C emissions tended to increase with distance from the glacier front. On average, the presence of vegetation increased CO2-C emissions by 440%, or the equivalent of 0.633g of CO2-C m(-2)h(-1). Results suggest that newly exposed landsurfaces undergo soil formation with increasing labile C input from vegetation, accompanied by increasing soil CO2-C emissions. Despite the importance of exposure time on CO2-C production and emissions, there was no similar trend in soil N2O-N production potentials as a function of glacial retreat. For N2O, instead, the maximum production occurred in sites with the first stages of vegetation growth.

Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-gamma as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi.

We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.

Structural studies on the polar glycoinositol phospholipids of Trypanosoma (Schizotrypanum) dionisii from bats.

The polar glycoinositol phospholipids (GIPLs) of a Trypanosoma species that belongs to the Schizotrypanum subgenus were purified by reversed-phase and normal-phase liquid chromatography and analysed by negative-ion mode electrospray-mass spectrometry (ES-MS). The phosphatidylinositol moieties were released by nitrous acid deamination and identified as ceramide- and alkylacylglycerol-containing species. The structures of the GIPLs were determined using chemical treatments, sequential exoglycosidase digestions and positive-ion mode ES-MS-MS. All of the GIPLs were based on the same Man alpha1-2Man alpha1-2Man alpha1-6Man alpha1-4(NH2-CH2CH2-HPO3-)GlcN-PI core with single terminal Galf residue substitutions either on the terminal nonreducing Man or on the second alphaMan residue from the inositol and with either ethanolamine phosphate or 2-aminoethylphosphonate on the third alphaMan residue from the inositol. The T. (S.) dionisii GIPLs are compared with those of T. (S.) cruzi, a closely related species of the Schizotrypanum subgenus.

The mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles.

Trypanosoma cruzi expresses at its surface large amounts of mucin-like glycoproteins. The T. cruzi mucins (TcMUC), a group of highly glycosylated GPI-anchored proteins rich in Thr, Ser, and Pro residues, are expressed in high copy numbers in both insect and mammalian stages of the parasite. These molecules are encoded by a multigene family and contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. The TcMUC are important because of their role in host cell invasion and the ability to induce secretion of proinflammatory cytokines and nitric oxide in activated macrophages. The TcMUC are also significant in being the major substrate for the cell surface trans-sialidase. In this review, we summarize the recent knowledge on the molecular structure and function of this family of T. cruzi glycoproteins.

Differential inhibitory mechanism of cyclic AMP on TNF-alpha and IL-12 synthesis by macrophages exposed to microbial stimuli.

Microbial stimuli such as bacterial lipopolysaccharide (LPS) or glycosylphosphatidylinositol-mucins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucins) are effective stimulators of the synthesis of cytokines by macrophages. Here, we evaluated the ability of cyclic AMP mimetic or elevating agents to modulate TNF-alpha and IL-12 synthesis by murine inflammatory macrophages. Cholera Toxin (ChTx) inhibited tGPI-mucins (2.5 nM) or LPS (100 ng ml(-1)) induced TNF-alpha and IL-12(p40) synthesis in a concentration-dependent manner. Similarly, the cyclic AMP mimetics, 8-bromo cyclic AMP or dibutyryl cyclic AMP, or prostaglandin (PG) E2 inhibited the synthesis of both cytokines by macrophages exposed to microbial stimuli. The protein kinase A inhibitor H-89 partially reversed the inhibitory effects of dibutyryl cyclic AMP and PGE2 on both IL-12(p40) and TNF-alpha synthesis. Pretreatment of macrophages with dibutyryl cyclic AMP or ChTx augmented the synthesis of IL-10 triggered by microbial products. Elevation of cyclic AMP inhibited the synthesis of TNF-alpha, but not IL-12(p40), by inflammatory macrophages from IL-10 knockout mice. Kinetic studies showed that synthesis of both TNF-alpha and IL-10 peaked at 8 h and IL-12 at 24 h after stimulation with microbial stimuli. Together, our findings favour the hypothesis that the cyclic AMP inhibitory activity on IL-12(p40) but not on TNF-alpha synthesis is dependent on de novo protein synthesis, most likely involving IL-10, by macrophages stimulated with microbial products. Accordingly, dibutyryl cyclic AMP inhibited IL-12(p40) synthesis only when added before or at the same time of the stimuli. In contrast, the effect of this cyclic AMP analogue on TNF-alpha synthesis was protracted and observed even 2 h after the addition of the stimuli.

On the biosynthesis of Rhodnius prolixus heme-binding protein.

The biosynthesis of Rhodnius prolixus heme-binding protein (RHBP), which is present in the hemolymph and oocytes of Rhodnius prolixus, was investigated. Fat bodies of female insects incubated in vitro with 14C-leucine were able to synthesize and secrete 14C-RHBP to the culture medium. Titrtion of synthesized RHBP with hemin showed that the protein secreted by the fat bodies is bound to heme, despite the presence of apo-RHBP in the hemolymph. The sequence of the RHBP cDNA encodes a pre-protein of 128 amino acids with no significant homology to any known protein. Northern-blot assays revealed that RHBP expression was limited to fat bodies. The levels of both RHBP mRNA and secreted protein increased in response to blood meal. In addition, the time-course of RHBP secretion in vitro paralleled mRNA accumulation observed in vivo. The inhibition of the de novo heme biosynthesis by treatment of fat bodies with succinyl acetone (SA), an irreversible inhibitor of delta-aminolevulinic acid-dehydratase, led to a significant decrease of heme-RHBP secretion. Nevertheless, the levels of RHBP mRNA were not modified by SA treatment, suggesting that the heme availability is involved in a post-transcriptional control of the RHBP synthesis.

Biochemistry and molecular biology of the main diagnostic antigen of Paracoccidioides brasiliensis.

The 43,000 dalton glycoprotein of Paracoccidioides brasiliensis (gp 43) is the main exocellular antigen recognized by sera from patients with paracoccidioidomycosis in a variety of serological assays. Specific conformational peptide epitopes are recognized by the human antibodies as determined by antigen deglycosylation. Procedures for the purification of the gp43 using immunoaffinity chromatography have been described. The secretion of the gp43 as a function of the growth curve, its partial aggregation with a proteolytic enzyme, ability to bind laminin, as well as to form circulating immunocomplexes in vivo could play a role in pathogenesis. Crude antigenic preparations depleted of gp43 epitopes lost their ability to elicit positive skin tests. Accordingly, the purified gp43 molecule induced delayed hypersensitivity reactions in man and infected animals, caused a T-CD4-dependent proliferation of lymph node cells from mice immunized with it, and of peripheral blood lymphocytes from an individual sensitized to P. brasiliensis by prolonged contact with the fungus. To identify the immunodominant epitopes in both humoral and cellular reactions, the gp43 gene has been cloned, sequenced, and partly expressed. It bears peptide sequences homologous to those of beta-1,3-glucanases from Candida albicans and Saccharomyces cerevisiae but has no enzymatic activity itself. The molecular weight of the unglycosylated antigen is 42,227. A single N-linked oligosaccharide chain in the gp43 contains alpha-D-mannopyranosyl, beta-D-galactofuranosyl and N-acetylglucosaminyl units with the predominant ratio of 10:2:2, and characteristics of a high mannose type.

GPI-anchored glycoconjugates from Trypanosoma cruzi trypomastigotes are recognized by lytic anti-alpha-galactosyl antibodies isolated from patients with chronic Chagas' disease.

The target molecules on the cell surface of Trypanosoma cruzi trypomastigotes reacting with lytic anti-alpha-galactosyl antibodies from chronic patients with Chagas' disease (Ch anti-Gal) have been purified by solvent extraction and identified as glycoconjugates migrating in the 74-96-kDa range (F2 antigen) and in the 120-200-kDa range (F3 antigen) on SDS-PAGE. The F3 antigen was tested for binding to Ch and normal human serum (NHS) anti-Gal and to MoAb 3C9. We observed that Ch anti-Gal and MoAb 3C9, but not NHS anti-Gal, bind strongly to the trypomastigote glycoconjugates. These antibodies, however, did not compete with each other for binding to F3 molecules, indicating that they are recognizing different epitopes. Binding of Ch anti-Gal to F3 antigen is abolished by treatment of these molecules with alpha- but not beta-galactosidase. Binding of 3C9 MoAb is abolished by treatment of F3 with sialidase. F2/F3 antigens absorbed Ch anti-Gal as well as lytic antibodies from total chagasic sera. These antigens also specifically discriminate between the serum reactivity of patients with active Chagas' disease and those of sera from cured patients, drug-treated patients with dissociated serology (positive conventional serology, negative trypanolytic activity), healthy individuals, and patients with several other infectious diseases. We also observed that F2/F3 antigens are anchored to the parasite membrane via glycosylphosphatidylinositol (GPI). The alpha-galactosyl epitopes recognized by Ch anti-Gal are present in a series of O-linked oligosaccharide chains in the mucin-like glycoprotein component of the complex.

[Prevalence, treatment needs, and predisposing factors for traumatic injuries to permanent dentition in 11-13-year-old schoolchildren]. / Prevalência, necessidade de tratamento e fatores predisponentes do traumatismo na dentição permanente de escolares de 11 a 13 anos de idade.

The objective of this study was to assess the prevalence of traumatic injuries to the permanent incisors and the association with clinical predisposing factors and parents' schooling. A cross-sectional survey was conducted with schoolchildren aged 11 to 13 years in Biguaçu, Brazil. Dental examinations were conducted by a dentist, and the criteria for traumatic dental injuries used in the children's dental health survey in the United Kingdom were adopted. the study recorded the type of damage sustained, treatment performed or needed, the size of incisal overjet, and whether lip coverage was adequate. Socio-demographic data included sex, age, and parents' level of schooling. a total of 2,260 children were examined, and prevalence rates were 10.4%, 10.6%, and 11.2% in 11, 12, and 13-year-old children, respectively. Treatment need was 6.3 interventions per thousand incisors. Male gender and overjet greater than 5mm were significantly related to having a traumatic dental injury. Inadequate lip coverage and parents' educational level were not associated with dental trauma. The study concluded that male gender and incisal overjet greater than 5mm are associated with the occurrence of dental injury.

High-content imaging for automated determination of host-cell infection rate by the intracellular parasite Trypanosoma cruzi.

Chagas disease affects 8-11 million people, mostly in Latin America. Sequelae include cardiac, peripheral nervous and/or gastrointestinal disorders, thus placing a large economic and social burden on endemic countries. The pathogenesis and the evolutive pattern of the disease are not fully clarified. Moreover, available drugs are partially effective and toxic, and there is no vaccine. Therefore, there is an urgent need to speed up basic and translational research in the field. Here, we applied automated high-content imaging to generate multiparametric data on a cell-by-cell basis to precisely and quickly determine several parameters associated with in vitro infection of host cell by Trypanosoma cruzi, the causative agent of Chagas disease. Automated and manual quantifications were used to determine the percentage of T. cruzi-infected cells in a 96-well microplate format and the data generated was statistically evaluated. Most importantly, this automated approach can be widely applied for discovery of potential drugs as well as molecular pathway elucidation not only in T. cruzi but also in other human intracellular pathogens.

Erosive effect of an antihistamine-containing syrup on primary enamel and its reduction by fluoride dentifrice.

AIM: This study evaluated the action of an antihistamine-containing syrup (Claritin D) on enamel that was subsequently submitted or not to applications of fluoride dentifrice. METHODS; Two hundred sixty-four slices (n=44 per subgroup) prepared from exfoliated primary molars were evaluated in hardness tests. Six subgroups were submitted to different treatments for 10 days. The controls underwent pH cycling with (positive control) or without (negative control) three daily immersions in fluoride dentifrice/distilled water slurry. The test subgroups related to daytime use of the antihistamine syrup underwent pH cycling and two 5-min applications of Claritin D, coupled or not to the three daily immersions in the fluoride slurry. The subgroups related to nocturnal use of the syrup were submitted to the same procedures of daytime subgroups, respectively, but with one of the applications of Claritin D lasting for 8 h. RESULTS: The median hardness values obtained after use of the syrup were significantly lower than the initial ones. Equivalent values for subgroups submitted to fluoride applications in addition to treatment with the syrup were significantly higher. CONCLUSION: It was concluded that the antihistamine-containing syrup reduced the hardness of primary enamel and that, in this experiment, the use of fluoride dentifrice was able to diminish this erosive effect.

Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains.

The aim of this study was to characterize a novel human autoantibody-autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3-20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine-tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization-mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease.

Chemiluminescent immunoassays: discrimination between the reactivities of natural and human patient antibodies with antigens from eukaryotic pathogens, Trypanosoma cruzi and Paracoccidioides brasiliensis.

Quantitative chemiluminescent enzyme-linked immunosorbent assay (ELISA) and dot-blotting procedures were developed to evaluate the reactivity of human antibodies with crude antigens and purified molecules of parasites and fungi, mainly Trypanosoma cruzi and Paracoccidioides brasiliensis. Reproducible, highly sensitive, and strictly dose-responding results were obtained, with the specificity depending on the kind of antigen used. Mixed antigens (epimastigote membrane and HIV-1 heptapeptide) applied in dots could be independently recognized by specific sera. Purified antigens (T. cruzi F2/3 and P. brasiliensis gp43) at very small concentrations gave specific reactions with patients' sera diluted > or = 1:1,000 and were very poorly reactive or unreactive with natural antibodies using the chemiluminescent immunoassays. P. brasiliensis crude antigen Fava Netto polysaccharide antigen (FNPA) contained peptide epitopes recognized by natural antibodies and carbohydrate epitopes reactive with sera from histoplasmosis patients. It is very important that sensitive chemiluminescence immunoassays be used with purified antigenic molecules to ensure specificity for the diagnosis and follow-up of parasitic and fungal infections.

Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi.

Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

Glycoconjugates of Trypanosoma cruzi: a 74 kD antigen of trypomastigotes specifically reacts with lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas disease.

Protective, lytic antibodies are believed to be correlated with active Trypanosoma cruzi infection. In patients with chronic infection, antibodies lysing trypomastigote forms recognize chiefly alpha-galactosyl structures at the parasite surface. The target molecules on cell-derived trypomastigotes that react with anti-alpha-galactosyl antibodies (anti-Gal) from patients with chronic Chagas disease were investigated. Glycoconjugates were isolated from trypomastigotes and shown to absorb purified Chagasic (Ch) anti-Gel effectively as well as lytic antibodies from Ch sera. Active fractions were F2 (74 kD and 95.6 kD) and F3 (120-200 kD). A differential reactivity with antibodies from untreated Ch patients (trypanolytic) and from treated, presumably cured, individuals (not trypanolytic) was evident using F2 and F3 antigenic fractions. No cross-reactivity with heterologous sera (other infections) was observed. The F2 glycoconjugate (mostly 74 kD) can be used in the diagnosis of active Chagas infection, replacing the quantitative determination of complement-mediated lysis. With the present sample of patients' sera and normal human sera, it showed 100% sensitivity and specificity.