Pectin is a complex carbohydrate soluble fiber. Dietary fibers, such as pectin, have been shown to have positive effects on a wide spectrum of pathological conditions. Their positive influence on human health is explained by their anti-oxidative, hypocholesterolemic, and anti-cancer effects [1
]. The effect on the immune system has been previously attributed to the down regulation of the inflammatory response, moderating the production of pro inflammatory cytokines and immunoglobulins in murine models for irritable bowel syndrome [13
]. A diet rich in soluble fiber in an animal model showed protection against endotoxin-induced sickness behavior by cytokine modulation and promotion of alternative activation of macrophages [14
]. Citrus pectin has the capacity to exert a favourable immunomodulatory response in human peripheral blood cells through its effect on cytokine production [15
]. High methoxy citrus pectin inhibits the binding of fibroblast growth factor-1 (FGF-1) to its receptor in the presence of heparin [16
]. The rhamnogalacturonan I-arabinan fraction of pectin from a medicinal herb enhances secretion of granulocyte colony-stimulating factor (G-CSF) by murine colonic MCE 301 cells [17
]. Rhamnogalacturonan I-arabinogalactan was also reported to activate macrophages and dendritic cells [18
]. Methyl-esterified pectic oligosaccharides with 4,5-unsaturated non-reducing ends enhanced T-helper1 (Th1) dependent delayed-type hypersensitivity in a murine influenza vaccine model, reduced Th2 cytokine (IL-4, IL-5 and IL-10) production in splenocytes in vitro
] and decreased allergic asthma in mice [20
]. Therefore, the carbohydrate composition of pectin is very important in determining different immune responses.
The modified citrus pectin (MCP) used in this study, is composed of short, slightly-branched, carbohydrate chains derived from the soluble albedo fraction of citrus fruit peels altered by decreasing the molecular weight and degree of esterification using pH, temperature and a controlled enzymatic process, in order to increase its absorption into the circulatory system. MCP is relatively rich in galactose, and antagonizes a binding protein galectin-3 (Gal-3), which results in suppression of cancer cell aggregation, adhesion, and metastasis [5
]. MCP acts as a ligand for Gal3, which plays a major role in tumor formation and progression [12
]. It has been shown using a combination of fluorescence microscopy, flow cytometry, and atomic force microscopy, that specific binding of a pectin galactan to the recombinant form of human galectin-3 has been physically observed [25
]. Moreover, MCP also showed anti-metastatic effects on cancer cells in vitro
or in vivo
]. Human clinical trials with MCP showed an increase in prostate specific antigen doubling time, a marker of slowing the progression of prostate cancer [9
], and significant improvement of quality of life and stabilization of disease for patients with advanced solid tumors [29
]. Besides the therapeutic roles against cancer, MCP has been shown to remove toxic metals from the body [30
], and reduce experimentally induced kidney injury and fibrosis in vivo
by reducing galectin-3 levels [32
]. In the United States of America, MCP is registered as a food supplement and is generally regarded as safe (GRAS).
lymphocyte activation represents a standard approach for evaluating cell-mediated responses to a variety of stimuli including immunostimulatory botanical extracts. An appropriate assay system monitors the expression of the early activation marker CD69 in whole blood after stimulation with extracts. CD69 is expressed in all activated lymphocyte subsets and hence it represents a generic marker to monitor individual subset responses to specific stimuli [33
]. CD4 antigen is expressed on the T- helper/inducer lymphocyte subset (CD3/CD4). CD8 antigen is expressed on the human cytotoxic T-lymphocyte subset (CD3/CD8). Once activated both CD4 and CD8 positive T cells express CD69. T-lymphocyte subsets can be identified and quantified by using fluorochrome-labelled antibody combinations such as CD4/CD69/CD3 and CD8/CD69/CD3. CD19 antigen is present on human B-lymphocytes at all stages of maturation and is not present on resting or activated T-lymphocytes. CD19/CD69/CD45 labelled antibody combination can be used to identify an activated B cell population. CD56 antigen is present on Natural Killer (NK)-cells and antigen intensity increases with NK-cell activation. Hence, CD56/CD69/CD45 labelled antibody combination can be used to identify activated NK-cells. The ability of NK-cell subset in normal lymphocytes to induce death in leukemia cells is analyzed by co-incubating MCP treated lymphocytes with K562 T-cell leukemia cells. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets (T-helper/inducer, T-cytotoxic, B-cell, and NK-cell), and the induced NK-cell's activity against K562 chronic myeloid leukemia cells. Carbohydrate composition analysis was also performed to propose a structural mechanism of action of this immune enhancement.