RESUMO
Air pollution is a serious environmental issue worldwide in developing countries' megacities, affecting the population's health, including the ocular surface, by predisposing or exacerbating other ocular diseases. Herpes simplex keratitis (HSK) is caused by the herpes simplex virus type 1 (HSV-1). The primary or recurring infection in the ocular site causes progressive corneal scarring that may result in visual impairment. The present study was designed to study the immunopathological changes of acute HSK under urban polluted air, using the acute HSK model combined with an experimental urban polluted air exposure from Buenos Aires City. We evaluated the corneal clinical outcomes, viral DNA and pro-inflammatory cytokines by RT-PCR and ELISA assays, respectively. Then, we determined the innate and adaptive immune responses in both cornea and local lymph nodes after HSV-1 corneal by immunofluorescence staining and flow cytometry. Our results showed that mice exposed to polluted air develop a severe form of HSK with increased corneal opacity, neovascularization, HSV-1 DNA and production of TNF-α, IL-1ß, IFN-γ, and CCL2. A high number of corneal resident immune cells, including activated dendritic cells, was observed in mice exposed to polluted air; with a further significant influx of bone marrow-derived cells including GR1+ cells (neutrophils and inflammatory monocytes), CD11c+ cells (dendritic cells), and CD3+ (T cells) during acute corneal HSK. Moreover, mice exposed to polluted air showed a predominant Th1 type T cell response over Tregs in local lymph nodes during acute HSK with decreased corneal Tregs. These findings provide strong evidence that urban polluted air might trigger a local imbalance of innate and adaptive immune responses that exacerbate HSK severity. Taking this study into account, urban air pollution should be considered a key factor in developing ocular inflammatory diseases.
Assuntos
Poluição do Ar/efeitos adversos , Exposição Ambiental/efeitos adversos , Ceratite Herpética/etiologia , Ceratite Herpética/patologia , Animais , Biomarcadores , Córnea/imunologia , Córnea/metabolismo , Córnea/patologia , Opacidade da Córnea/diagnóstico por imagem , Opacidade da Córnea/etiologia , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Imunofluorescência , Herpesvirus Humano 1 , Humanos , Imunofenotipagem , Ceratite Herpética/diagnóstico por imagem , Ceratite Herpética/metabolismo , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Lectins are glycan-binding proteins that are resistant to digestion in the gastrointestinal tract and enter intact to blood circulation. The aim of this study was to evaluate the influence of edible mushroom Agaricus bisporus lectin (ABL) on innate and adaptive immune responses as well as its effect in two different experimental pathologies that involve the immune system. ABL inhibited in vitro nitric oxide (NO) production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharides (LPS). However, it did not modify the activity of arginase, showing that while ABL downregulates M1 activation, it does not affect M2 activation. ABL also inhibited mononuclear cell proliferation in response to mitogen Con A, or in a mixed lymphocyte reaction. During the in vivo studies, oral administration of ABL to BALB/c mice induced a marked inhibition of NO production by peritoneal macrophages after LPS stimuli. The influence of ABL on tumor growth was studied in BALB/c mice receiving daily oral doses of ABL and implanted with CT26 tumor cells. ABL treatment induced significantly higher rate of tumor growth when compared with control mice. On the other hand, oral ABL administration in Wistar rats induced a marked diminution of the incidence of the disease and the severity of the clinical signs of experimental autoimmune encephalomyelitis. We can conclude that ABL has an in vivo immunomodulatory effect reducing the innate and adaptive responses. This food lectin shows potential therapeutic application on control of inflammatory autoimmune pathologies.
Assuntos
Fatores Imunológicos/farmacologia , Lectinas/farmacologia , Linfócitos/efeitos dos fármacos , Agaricus/química , Animais , Antineoplásicos , Proliferação de Células , Células Cultivadas , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Lectinas/química , Linfócitos/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Ratos , Ratos WistarRESUMO
Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type ("ricin-like") lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.
Assuntos
N-Acetilgalactosaminiltransferases/química , Polissacarídeos/química , Acetilação , Sequência de Aminoácidos , Catálise , Humanos , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/genética , Ligação Proteica , Conformação Proteica , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Cancer-associated mucins show frequent alterations of oligosaccharide chain profile. Terminal structures may be deleted, thereby exposing normally 'cryptic' structures such as Tn (GalNAcα-O-Ser/Thr) and T antigen (Galß1-3GalNAcα-O-Ser/Thr). Overexpression of these commonly hidden glycoforms, and reduced level of naturally occurring anti-T or anti-Tn antibodies, is associated with epithelial tumor progression and aggressiveness. The lectin from the common edible mushroom Agaricus bisporus (ABL) shows high affinity binding to T antigen, and reversible noncytotoxic inhibitory effect on epithelial tumor cell proliferation. The aim of this study was to induce immune response with tumor-associated glycan specificity and biological activity similar to those of ABL. An anti-idiotypic (Id) antibody strategy was developed using ABL as first template. ABL was purified by affinity chromatography and assayed as immunogen in rabbit. Rabbit IgG was purified from anti-ABL serum using a protein G column, and specific anti-ABL IgG was obtained by affinity chromatography using immobilized ABL. Affinity-purified anti-ABL IgG contained an antibody fraction that recognizes the carbohydrate-binding site of ABL. This IgG was used as immunogen in mouse to yield anti-Id antibody recognizing tumor-associated glycans such as Tn and T antigen. Competitive assays showed that α-anomeric GalNAc is the main binding subsite of anti-Id antibody in glycan recognition. Anti-Id antibody bound human epithelial tumor cells, as shown by cell enzyme-linked immunosorbent assay and immunofluorescence. Anti-Id antibody raised by immunization with affinity-purified anti-ABL IgG had antiproliferative effect on human epithelial tumor cells through apoptosis induction similar to that of ABL. The anti-Id immune response developed here has potential application in cancer therapy.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Carcinoma/imunologia , Lectinas/imunologia , Agaricus/imunologia , Animais , Antígenos Virais de Tumores/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/imunologia , Células Cultivadas , Cromatografia de Afinidade , Epitopos/metabolismo , Imunofluorescência , Humanos , Imunização , Imunoglobulina G/imunologia , Lectinas/isolamento & purificação , Lectinas/metabolismo , Camundongos , Ligação Proteica/imunologia , CoelhosRESUMO
Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes.
Assuntos
Antígenos de Neoplasias/farmacologia , Dissacarídeos/farmacologia , Epitopos de Linfócito T/farmacologia , Neoplasias Epiteliais e Glandulares/terapia , Vacinas Sintéticas/farmacologia , Animais , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Configuração de Carboidratos , Linhagem Celular Tumoral , Dissacarídeos/química , Dissacarídeos/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Epiteliais e Glandulares/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates.
Assuntos
Metabolismo dos Carboidratos , Euphorbia/química , Lectinas/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , EstereoisomerismoRESUMO
Cancer-associated mucins show frequent alterations of their oligosaccharide chain profile, with a switch to unmask normally cryptic O-glycan backbone and core regions. Epithelial tumour cells typically show overexpression of the uncovered Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr (Core 1) structure, known as the T antigen or the Thomsen-Friedenreich antigen, the oligosaccharide chain of which is called the Thomsen-Friedenreich disaccharide (TFD). T antigen expression has been associated with immunosuppression, metastasis dissemination, and the proliferation of cancer cells. Several different strategies have been used to trigger a specific immune response to TFD. Natural T antigen and synthetic TFD residues have low immunodominance. In the T antigen, flexibility of the glycosidic bond reduces the immunogenicity of the sugar residue. Enhanced rigidity should favour certain glycan conformations and thereby improve TFD immunotargeting. We propose the term 'glycan engineering' for this approach. Such engineering of TFD should reduce the flexibility of its glycan moiety and thereby enhance its stability, rigidity and immunogenicity.