Viana SM, Montoya AL, Carvalho AM, de Mendonça BS, Portillo S, et al.
Emerging microbes & infections •
American tegumentary leishmaniasis (TL) caused by is characterized by a spectrum of clinical presentations, ranging from localized cutaneous ulcers (CL), mucosal (ML), or disseminated (DL) disease, to a subclinical (SC) asymptomatic form. Current diagnosis based on parasite culture and/or microscopy lacks sensitivity and specificity. Previous studies showed that patients with CL and ML have very high levels of -specific anti-α-Gal antibodies. However, the native parasite α-Gal glycotope(s) is(are) still elusive, thus they have not yet been explored for a more accurate TL diagnosis. Using a chemiluminescent immunoassay, we evaluated the seroreactivity of TL patients across its clinical spectrum, and of endemic (EC) and nonendemic healthy controls (NEC) against three synthetic neoglycoproteins (NGP29b, NGP30b, and NGP28b), respectively comprising the derived type-2 glycoinositolphospholipid (GIPL)-1 (Galβ1,3Manα), GIPL-2 (Galα1,3Galβ1,3Manα), and GIPL-3 (Galα1,6Galα1,3Galβ) glycotopes. Contrary to NGP29b and NGP30b, NGP28b exhibited high sensitivity and specificity to a CL serum pool. More importantly, NGP28b reacted strongly and specifically with individual sera from distinct clinical forms of TL, especially with SC sera, with 94% sensitivity and 97% specificity, by post-two-graph receiver-operating characteristic curve analysis. Contrary to NGP29b, NGP28b showed low cross-reactivity with Chagas disease and control (NEC/EC) sera. Additionally, seroreactivity of CL patients against NGP28b was significantly decreased after successful chemotherapy, indicating that -specific anti-α-Gal antibodies may serve as an early biomarker of cure in CL. Our data also points towards the applicability of type-2 GIPL-3-derived Galα1,6Galα1,3Galβ glycotope for the serological diagnosis of American TL, particularly of the subclinical form.
Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.
Gazzinelli RT, Galvao LM, Krautz G, Lima PC, Cancado JR, et al.
The American journal of tropical medicine and hygiene •
With the exception of assays for the detection of antibodies promoting complement-mediated lysis of Trypanosoma cruzi, serologic tests have generally failed to assess the effectiveness of chemotherapy for Chagas' disease. Conventional serology, although useful for the diagnosis of infection, is not capable of determining which patients have been cured. Here we demonstrate that a high proportion of antibodies detected by conventional serology (using fixed epimastigotes or trypomastigotes or crude extracts obtained therefrom) are directed against the carbohydrate residue galactosyl alpha 1- > 3 galactose (Gal alpha 1- > 3 Gal), a determinant also recognized by antibodies from noninfected healthy volunteers. In a study of 14 cured patients with long-term followup, we found that the persistently positive reactions detected using conventional serology were largely eliminated following immunoadsorption with melibiose. Because of their wide distribution among microorganisms of intestinal and pulmonary microflora, these carbohydrate determinants may keep stimulating lymphocytes previously primed by T. cruzi Gal alpha 1- > 3 Gal epitopes, thereby accounting for false-positive results in cured patients. Consistent with this proposition, enzyme-linked immunosorbent assays performed with two distinct T. cruzi antigen preparations that lack the Gal alpha 1- > 3 Gal epitope, namely purified GP57/51 and trypomastigote-shed antigens, were indeed capable of determining a cure after chemotherapy, albeit to a different degree. Collectively, the data indicate that conventional immunoassays prepared with highly specific T. cruzi antigens can be useful in the assessment of a cure after chemotherapy.
Gazzinelli RT, Pereira ME, Romanha A, Gazzinelli G, Brener Z
Parasite immunology •
Anti-gal antibodies directed against a carbohydrate epitope present in mouse laminin (galactosyl alpha 1-3 galactose) and detected in high levels in sera from patients in the acute phase of Chagas disease are responsible for the direct lysis (DL) of Trypanosoma cruzi blood forms independent of either the classic or alternative complement pathways. Furthermore, the lectins Euonymus europaeus (EE) specific for the carbohydrates gal alpha 1-3 gal present a similar lytic activity against T. cruzi at the same concentrations of purified anti-gal antibodies. The DL activity was tested with several other lectins but Concanavalin A (Con A) specific for alpha-D-mannose and alpha-D-glucose was the only one also presenting lytic activity. The lectins and anti-gal antibodies lytic activity can be inhibited by specific carbohydrates suggesting that this phenomenon is related to the capability of these lectins or anti-gal antibodies to bind to a crucial surface component of T. cruzi. Moreover, the infectivity of T. cruzi blood forms to mice was clearly inactivated by incubation with acute chagasic sera (ACS) but not by ACS absorbed by immunoaffinity chromatography with mouse laminin, a strong evidence that high levels of anti-gal antibodies participate in the decline of the parasitaemia from the acute to the chronic phase in Chagas disease.
Gazzinelli RT, Romanha AJ, Fontes G, Chiari E, Gazzinelli G, et al.
The Journal of protozoology •
We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.
Gazzinelli R, Galvão LM, Dias JC, Gazzinelli G, Brener Z
Transactions of the Royal Society of Tropical Medicine and Hygiene •
A striking increase of anti-laminin antibodies contrasting with low levels of anti-Trypanosoma cruzi antibodies was detected by enzyme-linked immunosorbent assay in sera of 16 patients at the acute phase of Chagas disease. The mean absorbancies using an anti-human IgG conjugate were 6563 and 202 for laminin and T. cruzi antigens, respectively; with anti-IgM conjugate the titres were 3442 and 207, respectively. In addition, indirect immunofluorescence tests performed with trypomastigotes from blood or cell cultures have shown extremely high titres (up to 1:10 240), which declined when mouse laminin-absorbed sera were used instead, apparently confirming the presence of laminin in the surface membrane of the parasites. These results support the view that the high anti-laminin titres found in patients with Chagas disease are induced by the newly described galactosyl-alpha 1-3-galactose epitope present in T. cruzi trypomastigotes and shared by mouse laminin.
