RESUMO
Neglected tropical diseases, such as Chagas disease caused by the protozoa Trypanosoma cruzi, affect millions of people worldwide but lack effective treatments that are accessible to the entire population, especially patients with the debilitating chronic phase. The recognition of host cells, invasion and its intracellular replicative success are essential stages for progression of the parasite life cycle and the development of Chagas disease. It is predicted that programmed cell death pathways (apoptosis) would be activated in infected cells, either via autocrine secretion or mediated by cytotoxic immune cells. This process should play a key role in resolving infections by hindering the evolutionary success of the parasite. In this research, we performed assays to investigate the role of the lectin galectin-3 (Gal3) in parasite-host signaling pathways. Using cells with endogenous levels of Gal3 compared to Gal3-deficient cells (induced by RNA interference), we demonstrated that T. cruzi mediated the survival pathways and the subverted apoptosis through Gal3 promoting a pro-survival state in infected cells. Infected Gal3-depleted cells showed increased activation of caspase 3 and pro-apoptotic targets, such as poly (ADP-ribose) polymerase (PARP), and lower accumulation of anti-apoptotic proteins, such as c-IAP1, survivin and XIAP. During the early stages of infection, Gal3 translocates from the cytoplasm to the nucleus and must act in survival pathways. In a murine model of experimental infection, Gal3 knockout macrophages showed lower infectivity and viability. In vivo infection revealed a lower parasitemia and longer survival and an increased spleen cellularity in Gal3 knockout mice with consequences on the percentage of T lymphocytes (CD4+ CD11b+) and macrophages. In addition, cytokines such as IL-2, IL-4, IL-6 and TNF-α are increased in Gal3 knockout mice when compared to wild type genotype. These data demonstrate a Gal3-mediated complex interplay in the host cell, keeping infected cells alive long enough for infection and intracellular proliferation of new parasites. However, a continuous knowledge of these signaling pathways should contribute to a better understanding the mechanisms of cell death subversion that are promoted by protozoans in the pathophysiology of neglected diseases such as Chagas disease.
Assuntos
Apoptose/fisiologia , Doença de Chagas/parasitologia , Galectina 3/fisiologia , Trypanosoma cruzi/fisiologia , Análise de Variância , Animais , Western Blotting , Caspase 3/análise , Sobrevivência Celular , Doença de Chagas/mortalidade , Chlorocebus aethiops , Colorimetria , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Galectina 3/análise , Galectina 3/genética , Células HeLa , Humanos , Imunofenotipagem , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parasitemia/mortalidade , Parasitemia/parasitologia , Fenótipo , Baço/patologia , Células VeroRESUMO
OBJECTIVE: To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients. METHODS: Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density. RESULTS: Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups. CONCLUSION: The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.
Assuntos
Anticorpos Monoclonais/análise , Doença de Chagas/patologia , Colonoscopia/métodos , Galectina 3/análise , Mucosa Intestinal/patologia , Megacolo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Biópsia , Estudos de Casos e Controles , Contagem de Células , Colágeno/análise , Feminino , Fibrose , Galectina 3/imunologia , Humanos , Imuno-Histoquímica , Masculino , Mastócitos/patologia , Pessoa de Meia-Idade , Miosite/patologia , Estudos Retrospectivos , Estatísticas não ParamétricasRESUMO
Galectin-3 (Gal-3) is a multifunctional glycan-binding protein that participates in many pathophysiological events and has been described as a biomarker and potential therapeutic target for severe disorders, such as cancer. Several probes for Gal-3 or its ligands have been developed, however both the pathophysiological mechanisms and potential biomedical applications of Gal-3 remain not fully assessed. Molecular imaging using bioluminescent probes provides great sensitivity for in vivo and in vitro analysis for both cellular and whole multicellular organism tracking and target detection. Here, we engineered a chimeric molecule consisting of Renilla luciferase fused with mouse Gal-3 (RLuc-mGal-3). RLuc-mGal-3 preparation was highly homogenous, soluble, active, and has molecular mass of 65,870.95â¯Da. This molecule was able to bind to MKN45â¯cell surface, property which was inhibited by the reduction of Gal-3 ligands on the cell surface by the overexpression of ST6GalNAc-I. In order to obtain an efficient and stable delivery system, RLuc-mGal-3 was adsorbed to poly-lactic acid nanoparticles, which increased binding to MKN45â¯cells in vitro. Furthermore, bioluminescence imaging showed that RLuc-mGal-3 was able to indicate the presence of implanted tumor in mice, event drastically inhibited by the presence of lactose. This novel bioluminescent chimeric molecule offers a safe and highly sensitive alternative to fluorescent and radiolabeled probes with potential application in biomedical research for a better understanding of the distribution and fate of Gal-3 and its ligands in vitro and in vivo.
Assuntos
Galectina 3/metabolismo , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/metabolismo , Neoplasias/diagnóstico por imagem , Polissacarídeos/metabolismo , Animais , Linhagem Celular Tumoral , Galectina 3/análise , Galectina 3/genética , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Substâncias Luminescentes/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Imagem Óptica , Polissacarídeos/análise , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
ABSTRACT Objective To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients. Methods Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density. Results Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups. Conclusion The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.
RESUMO Objetivo Avaliar a densidade de células imunomarcadas por anti-galectina-3, a percentagem de colágeno, a densidade de mastócitos e a presença de processos patológicos na musculatura intestinal de pacientes biopsiados. Métodos Foram selecionados 35 pacientes submetidos à biópsia de intestino entre 1997 a 2015. Os pacientes foram divididos em três grupos: chagásicos com lesão de mucosa (n=13), chagásicos com mucosa íntegra (n=12) e não chagásicos sem lesão de mucosa (n=10). Foram realizados processamento histológico dos fragmentos biopsiados e imunohistoquímica para galectina-3. Cortes adicionais foram corados por hematoxilina e eosina, para avaliar os processos patológicos gerais, pelo picrosírius, para avaliação do colágeno, e pelo azul de toluidina, para avaliar a densidade de mastócitos. Resultados Os pacientes do grupo chagásicos com lesão de mucosa apresentaram frequência significativamente maior de ganglionite e miosite quando comparados aos dos grupos chagásico com mucosa íntegra e não chagásicos. A densidade das células imunomarcadas por anti-galectina-3 foi significativamente maior no grupo chagásicos com mucosa íntegra quando comparada ao grupo não chagásico. O grupo de chagásicos com mucosa íntegra apresentou maior percentagem de colágeno em relação aos grupos chagásicos com mucosa lesada e ao grupo de não chagásicos, com diferença significativa. Não houve diferença significativa com relação à densidade de mastócitos entre os três grupos. Conclusão A maior densidade de células imunomarcadas por anti-galectina-3 nos pacientes do grupo chagásico com mucosa íntegra sugere a necessidade de maior atenção na avaliação clínica desses pacientes, uma vez que essa proteína está associada com transformação e progressão neoplásica.
Assuntos
Humanos , Masculino , Feminino , Adulto , Idoso , Idoso de 80 Anos ou mais , Colonoscopia/métodos , Doença de Chagas/patologia , Galectina 3/análise , Mucosa Intestinal/patologia , Megacolo/patologia , Anticorpos Monoclonais/análise , Biópsia , Fibrose , Imuno-Histoquímica , Estudos de Casos e Controles , Contagem de Células , Estudos Retrospectivos , Análise de Variância , Colágeno/análise , Estatísticas não Paramétricas , Galectina 3/imunologia , Mastócitos/patologia , Pessoa de Meia-Idade , Miosite/patologiaRESUMO
Malignant melanomas (MMs) represent 7% of all malignant neoplasms in dogs. Oral melanocytic neoplasms are often malignant and associated with poor prognosis. There are no universally accepted prognostic markers for canine oral melanoma. Galectin (Gal)-3 is a prognostic marker for human neoplasms such as thyroid, gastric, colorectal and prostate cancers. The protein is related to processes that favour cancer progression, such as angiogenesis, proliferation and apoptosis. The aim of the present study was to characterize the immunohistochemical expression of Gal-3 in canine oral melanomas and to compare it with post-surgical survival, the expression of apoptosis-related proteins and other known prognostic tools. Twenty-seven samples of canine oral melanomas were evaluated for Gal-3, B-cell lymphoma (BCL) 2, caspase (CASP) 3 and Ki67 expression, mitotic index and degree of nuclear atypia. Gal-3 cytoplasmic positivity was correlated positively, while nuclear positivity was correlated negatively, with survival. The intensity of BCL2 labelling was also correlated positively with Gal-3 cytoplasmic positivity. Higher nuclear atypia was observed in dogs with melanoma that died due to the tumour, as well as in dogs that survived for <1 year after surgery. We have confirmed the importance of nuclear atypia for MMs and suggest that Gal-3 is a valuable prognostic indicator for this neoplasm. More in-depth studies are needed to unveil Gal-3 functions in canine MMs using larger sample sizes.
Assuntos
Biomarcadores Tumorais/metabolismo , Doenças do Cão , Galectina 3/metabolismo , Melanoma/veterinária , Neoplasias Bucais/veterinária , Animais , Biomarcadores Tumorais/análise , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Galectina 3/análiseRESUMO
Keloids are defined histopathologically as an inflammatory disorder characterized by exhibiting numerous fibroblasts, abnormal vascularization, increased number of proinflammatory immune cells as well as uncontrolled cell proliferation, and exacerbated and disorganized deposition of extracellular matrix (ECM) molecules. Importantly, many of these ECM molecules display N- and O-linked glycan residues and are considered as potential targets for galectin-1 (Gal-1) and galectin-3 (Gal-3). Nevertheless, the presence and localization of Gal-1 and Gal-3 as well as the interactions with some of their binding partners in keloid tissues have not been considered. Here, we show that in the dermal thickening of keloids, versican, syndecan-1, fibronectin, thrombospondin-1, tenascin C, CD44, integrin ß1, and N-cadherin were immunolocalized in the elongated fibroblasts that were close to the immune cell infiltrate, attached to collagen bundles, and around the microvasculature and in some immune cells. We also show that Gal-1 and Gal-3 were present in the cytoplasm and along the cell membrane of some fibroblasts and immune and endothelial cells of the dermal thickening. We suggest that Gal-1 and Gal-3, in concert with some of the ECM molecules produced by fibroblasts and by immune cells, counteract the inflammatory response in keloids. We also proposed that Gal-1 and Gal-3 through their binding partners may form a supramolecular structure at the cell surface of fibroblasts, immune cells, endothelial cells, and in the extracellular space that might influence the fibroblast morphology, adhesion, proliferation, migration, and survival as well as the inflammatory responses.
Assuntos
Derme/química , Fibroblastos/química , Galectina 1/análise , Galectina 3/análise , Queloide/metabolismo , Adolescente , Adulto , Biomarcadores/análise , Proteínas Sanguíneas , Derme/patologia , Feminino , Fibroblastos/patologia , Galectinas , Humanos , Imuno-Histoquímica , Queloide/patologia , Masculino , Ligação Proteica , Adulto JovemRESUMO
Galectin-3 (Gal-3) is a protein expressed by both normal and neoplastic cells. It participates in several biological processes such as cell proliferation, cell adhesion, apoptosis, tissue remodeling, and angiogenesis. Although it is known to serve as a valuable prognostic marker in several types of human cancer, there are few reports about its applicability as a marker in the veterinary oncology literature. The aim of the present study was to characterize Gal-3 expression in different types of canine tumors. Fifty-three tissue samples from 22 histologically different types of canine tumors were immunohistochemically evaluated for Gal-3 expression. Variations in the percentage of Gal-3-positive cells, localization of Gal-3 protein, and percentage of Gal-3-positive fibroblasts were observed. These preliminary results showed variable expression of Gal-3 among canine tumors. Further studies are needed in order to investigate the potential of this protein as a prognostic marker and a therapeutic target.(AU)
Assuntos
Animais , Cães , Galectina 3/análise , Imuno-Histoquímica , Proteínas de Neoplasias/análise , Prognóstico , Neoplasias/veterinária , Neoplasias/diagnósticoRESUMO
OBJECTIVES: Galectins are mediators that play an important role in the inflammatory response and in this study we analyzed the expression of Galectins (Gal) -1, -3 and -9 in biopsies of the gastric antrum of patients with upper gastrointestinal symptoms. METHODOLOGY: 44 patients with upper digestive tract symptoms were evaluated, and underwent Upper Digestive Endoscopy examination. Sections of the gastric antrum were fixed in buffered formaldehyde at 4% in order to perform the anatomopathological examination and immunohistochemical analysis for Galectins-1, -3 and -9 expression. Fresh sections of gastric antrum were used for DNA extraction and evaluation of Helicobacter pylori (H. pylori). P values<0.05 were considered statistically significant. RESULTS: Gal-1 was significantly more expressed on stroma than epithelium (p<0.0001), whereas Gal-3 and Gal-9 were more expressed on epithelium (p<0.0001). Gal-3 was found to be significantly higher in the stroma of patients with H. pylori infection, mainly on Cag-A positive H. pylori (p<0.0001). Gal-9 was down modulated in stroma of patients with chronic gastritis. CONCLUSION: Up modulation of Gal-3 expression was associated with H. pylori infection and down modulation of Gal-9 with the inflammatory process of chronic gastritis.
Assuntos
Células Epiteliais/química , Galectina 3/análise , Galectinas/análise , Mucosa Gástrica/química , Gastrite/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/isolamento & purificação , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Biópsia , Proteínas Sanguíneas , Doença Crônica , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Imuno-Histoquímica , Células Estromais/química , Células Estromais/microbiologia , Células Estromais/patologiaRESUMO
OBJECTIVE AND DESIGN: The aim of the present study was to evaluate the immunohistochemical expression of Gal-1, Gal-3 and Gal-9 in the colon of chronic chagasic patients compared to biopsied non-chagasic patients. MATERIAL OR SUBJECTS: Thirty-two colon fragments were selected from chagasic patients with megacolon (n=25) and nonchagasic patients without megacolon (n=7). METHODS: Immunohistochemistry for Gal-1, Gal-3 and Gal-9 was performed using a common light microscope and the results were scored 0-3 according to labeling intensity. Data were analyzed statistically by the chi-square test. RESULTS: Higher Gal-1, Gal-3 and Gal-9 expression was observed in the myenteric plexus ganglia of chagasic patients compared to non-chagasic patients, p=0.0487, p=0.0019 and p=0.0325, respectively, whereas no significant differences were observed between groups regarding the expression of Gal-1, Gal-3 and Gal-9 in the muscle layer. CONCLUSION: Since Gal-1, Gal-3 and Gal-9 galectin expression was higher in the myenteric plexus ganglia of chagasic patients, we believe that these lectins may be associated with ganglionitis in the chagasic megacolon. However, since the present study was the first to report the participation of Gal-9 in Chagas disease, further investigations are needed to elucidate the role of galectin 9 in this disease.
Assuntos
Doença de Chagas/patologia , Galectina 1/biossíntese , Galectina 3/biossíntese , Galectinas/biossíntese , Idoso , Biomarcadores/análise , Proteínas Sanguíneas , Feminino , Galectina 1/análise , Galectina 3/análise , Galectinas/análise , Humanos , Imuno-Histoquímica , Masculino , Megacolo/microbiologia , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Macrophages are a functionally heterogeneous cell population and depending on microenvironments they polarize in two main groups: M1 and M2. Glutamic acid and glutamate receptors may participate in the regulation of macrophage plasticity. To investigate the role of glutamatergic systems in macrophages physiology, we performed the transfection of mGluR5 cDNAs into RAW-264.7 cells. RESULTS: Comparative analysis of modified (RAW-mGluR5 macrophages) and non-modified macrophages (RAW-macrophages) has shown that the RAW-mGluR5 macrophages absorbed more glutamate than control cells and the amount of intracellular glutamate correlated with the expression of excitatory amino acid transporters -2 (EAAT-2). Besides, our results have shown that RAW-mGluR5 macrophages expressed a higher level of peroxisome proliferator-activated receptor γ (PPAR-γ) and secreted more IL-10, high mobility group box 1 proteins (HMGB1) and Galectin-3 than control RAW-macrophages. CONCLUSIONS: We propose that elevation of intracellular glutamate and expression of mGluR5 may initiate the metabolic rearrangement in macrophages that could contribute to the formation of an immunosuppressive phenotype.