RESUMO
Lectin-carbohydrate binding may be involved in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria; therefore, we tested if this interaction is associated with snail resistance against Schistosoma infection. In vitro data showed that most of the S. mansoni sporocysts cultured with haemocytes from Biomphalaria glabrata BH, a highly susceptible snail strain, had a low number of cells that adhered to their tegument and a low mortality rate. Moreover, the addition of N-acetyl-D-glucosamine (GlcNAc) did not alter this pattern of adherence and mortality. Using haemocytes and haemolymph of Biomphalaria tenagophila Cabo Frio, we observed a high percentage of sporocysts with adherent cells, but complete encapsulation was not detected. Low concentrations of GlcNAc increased haemocyte binding to the sporocysts and mortality, which returned to basal levels with high concentrations of the carbohydrate. In contrast, haemocytes plus haemolymph from B. tenagophila Taim encapsulated cellular adhesion index of level 3 and destroyed over 30% of the S. mansoni sporocysts in culture. Interestingly, the addition of GlcNAc, but not mannose, to the culture medium resulted in the significant inhibition of cellular adhesion to the parasite tegument and the reduction of parasite mortality, suggesting that GlcNAc carbohydrate moieties are important to the recognition of S. mansoni by B. tenagophila Taim.
Assuntos
Acetilglucosamina/imunologia , Biomphalaria/parasitologia , Hemócitos/parasitologia , Hemolinfa/parasitologia , Oocistos/fisiologia , Schistosoma mansoni/imunologia , Animais , Biomphalaria/citologia , Carboidratos/imunologia , Interações Hospedeiro-ParasitaRESUMO
Lectin-carbohydrate binding may be involved in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria; therefore, we tested if this interaction is associated with snail resistance against Schistosoma infection. In vitro data showed that most of the S. mansoni sporocysts cultured with haemocytes from Biomphalaria glabrata BH, a highly susceptible snail strain, had a low number of cells that adhered to their tegument and a low mortality rate. Moreover, the addition of N-acetyl-D-glucosamine (GlcNAc) did not alter this pattern of adherence and mortality. Using haemocytes and haemolymph of Biomphalaria tenagophila Cabo Frio, we observed a high percentage of sporocysts with adherent cells, but complete encapsulation was not detected. Low concentrations of GlcNAc increased haemocyte binding to the sporocysts and mortality, which returned to basal levels with high concentrations of the carbohydrate. In contrast, haemocytes plus haemolymph from B. tenagophila Taim encapsulated cellular adhesion index of level 3 and destroyed over 30 percent of the S. mansoni sporocysts in culture. Interestingly, the addition of GlcNAc, but not mannose, to the culture medium resulted in the significant inhibition of cellular adhesion to the parasite tegument and the reduction of parasite mortality, suggesting that GlcNAc carbohydrate moieties are important to the recognition of S. mansoni by B. tenagophila Taim.
Assuntos
Animais , Acetilglucosamina/imunologia , Biomphalaria/parasitologia , Hemócitos/parasitologia , Hemolinfa/parasitologia , Oocistos/fisiologia , Schistosoma mansoni/imunologia , Biomphalaria/citologia , Carboidratos/imunologia , Interações Hospedeiro-ParasitaRESUMO
Single units of O-linked N-acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C-terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self-antigens like myosin are associated with Chagas heart disease. The participation of O-GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross-reactivity between Cz and myosin is mainly focused in the C-T domain. ELISA inhibition assays using rabbit sera specific for Cz and C-T in conjunction with immune-gold electron microscopy analysis of heart tissues from mice immunized with C-T confronted with polyclonal rabbit sera specific for Cz and C-T prior and after myosin adsorption provided evidence which indicates that O-GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O-GlcNAc-containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease.
Assuntos
Acetilglucosamina/imunologia , Anticorpos Antiprotozoários/imunologia , Reações Cruzadas , Cisteína Endopeptidases/imunologia , Epitopos/imunologia , Miosinas/imunologia , Trypanosoma cruzi/imunologia , Acetilglucosamina/análise , Animais , Cisteína Endopeptidases/química , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Miocárdio/patologia , Miosinas/química , Proteínas de Protozoários , Coelhos , Trypanosoma cruzi/químicaRESUMO
The GlcNAc-specific adhesin from Mannheimia haemolytica (MhA) has been shown to participate in pathogenicity of mannheimiosis due to its capacity to adhere to tracheal epithelial cells and activate the oxidative burst of bovine neutrophils. In this work, we purified the MhA receptor from bovine neutrophils (MhAr) by affinity chromatography on MhA-Sepharose. The MhAr, which corresponded to approximately 2% of the protein from cell lysate, is a glycoprotein mainly composed of Glu, Ala, Ser, Gly, and Asp, without cysteine. The glycan portion, which corresponds to 20% by weight, is composed of GalNAc, GlcNAc, Man, Gal, and NeuAc. The receptor is a 165-kDa glycoprotein, as determined by molecular sieve chromatography under native conditions; SDS-PAGE analysis shows a heterodimer of 83 and 80 kDa subunits. This work suggests that the GlcNAc-containing receptor plays a relevant role by activating bovine neutrophils through non-opsonic mechanisms.
Assuntos
Acetilglucosamina/metabolismo , Adesinas Bacterianas/metabolismo , Mannheimia haemolytica/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/isolamento & purificação , Acetilglucosamina/imunologia , Adesinas Bacterianas/imunologia , Animais , Bovinos , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Ativação de Neutrófilo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Explosão RespiratóriaRESUMO
In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.
Assuntos
Adesinas Bacterianas/farmacologia , Doenças dos Bovinos/microbiologia , Bovinos/sangue , Mannheimia haemolytica/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Pasteurelose Pneumônica/imunologia , Explosão Respiratória/efeitos dos fármacos , Acetilglucosamina/imunologia , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/isolamento & purificação , Animais , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Citometria de Fluxo/veterinária , Imuno-Histoquímica/veterinária , Iodoacetamida/farmacologia , Mannheimia haemolytica/isolamento & purificação , Ativação de Neutrófilo/imunologia , Neutrófilos/microbiologia , Pasteurelose Pneumônica/microbiologia , Piroxicam/farmacologia , Explosão Respiratória/imunologia , Superóxido Dismutase/farmacologiaRESUMO
We have previously reported the finding of circulating antibodies recognizing two proteins of 100 and 120 kD (PO100 and PO120) from Pityrosporum ovale in patients with psoriasis. These antibodies were specific, since they were not detected in normal sera nor in other diseases linked to P. ovale such as seborrhoeic dermatitis or pityriasis versicolor. The present study aimed at further characterizing the specificity of these antibodies. Enzyme-labelled lectins were used to determine the carbohydrate composition of PO120 and PO100. BSII, a lectin that recognizes terminal N-acetylglucosamine (GlcNAc), showed the same banding pattern as sera from patients. Reactivity against these proteins was inhibited after mild oxidation of the carbohydrate moieties of the extract. Treatment of the extracts with lyticase altered the immune reactivity against the PO120 band as seen in Western blot assays. PO100 was not detected after lyticase digestion. Digestion with lysozyme did not alter the immune reactivity of the PO100 and PO120 bands, although the protein pattern in SDS-PAGE was modified. To examine the relevance of anti-GlcNAc antibodies in the immune response to P. ovale in psoriasis, we performed a binding inhibition ELISA. Psoriatic sera that were positive in the ELISA against a heat-denatured extract of P. ovale were rendered negative only by pre-incubation with GlcNAc in a concentration-dependent manner. Our results are indicative that the antibody response to PO100 and PO120 in patients with psoriasis is directed towards terminal GlcNAc residues.