ABSTRACT
Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-D: -mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.
Subject(s)
Breast Neoplasms/metabolism , Carbohydrates/analysis , Luminescent Measurements/methods , Biomarkers/chemistry , Biomarkers/metabolism , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Carbohydrate Metabolism , Carbohydrates/chemistry , Carcinoma, Ductal, Breast/diagnosis , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Ductal, Breast/pathology , Concanavalin A/metabolism , Female , Fibroadenoma/diagnosis , Fibroadenoma/metabolism , Fibroadenoma/pathology , Humans , Middle Aged , Peanut Agglutinin/metabolismABSTRACT
Cramoll 1,4 is a lectin extracted from Cratylia mollis Mart. seeds that has shown antitumor and lymphocyte mitogenic activities in other studies. The aim of this work was to investigate, in vitro, the immunomodulatory activity of Cramoll 1,4 on experimental cultures of mice lymphocytes through cytotoxic assays, nitric oxide (NO) concentrations and IL-10 and IFN-γ production. Cramoll 1,4 did not show cytotoxic activity at 1-25 µg/mL concentrations, similar results were observed with concanavalin A (Con A) and phytohemagglutinin (PHA) lectins. The minimum production of IL-10 was observed in splenocytes cultivated with Con A, PHA and Cramoll 1,4 lectins. However, splenocytes treated with Cramoll 1,4 showed higher IFN-γ production in comparison with PHA and Con A (p < 0.05 for both). Production of NO was effectively suppressed in murine cells stimulated with the lectins and was only detected after 72 h for PHA in relation to non-stimulated lymphocytes (p < 0.05). Cramoll 1,4 was not toxic to murine lymphocytes, induced Th1 response through IFN-γ production and showed antiinflammatory activity through NO suppression. Therefore, Cramoll 1,4 can be considered a lectin with immunomodulatory activity.
Subject(s)
Fabaceae/chemistry , Lectins/pharmacology , Lymphocytes/drug effects , Animals , Cells, Cultured , Concanavalin A/pharmacology , Immunomodulation , Interferon-gamma/immunology , Interleukin-10/immunology , Lymphocytes/immunology , Male , Mice , Mice, Inbred BALB C , Nitric Oxide/metabolism , Phytohemagglutinins/pharmacology , Seeds/chemistry , Spleen/cytologyABSTRACT
This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 microg ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.
