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Title:   Author  item
                                            RAMACHANDRAN, CHEPPAIL - Dharma Biomedicalitem
                                            WILK, BARRY - Econugenics, Incitem
                                            Hotchkiss, Arlanditem
                                            Chau, Hoa

                                                         - *Rose*item
                                            ELIAZ, ISAAC - Econugenics, Incitem
                                            MELNICK, STEVEN - Miami Children'S Hospital
  Submitted to: BMC Complementary and Alternative Medicine
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/4/2011
                                    Publication  Date: 8/4/2011
                                    Citation: Ramachandran, C., Wilk, B.J., Hotchkiss, A.T., Chau, H.K., Eliaz, I., Melnick, S.J. 2011. Activation of human T-helper/inducer cell, T-cytotoxic/suppressor cell, B-cell, and natural killer (NK)-cells and induction of NK cell activity against K562 chronic myeloid leukemia cells with modified citrus pectin. BMC Complementary and Alternative Medicine. 11:59.Interpretive Summary: Agricultural residues remaining after fruit and vegetable processing, such as citrus peel and sugar beet pulp, represent an underutilized domestic resource of valuable bioactive food ingredients.  These residues have been used as cattle feed ingredients, but as such their value is low (under $0.05/pound) and there is often more supply than demand for this use.  We demonstrated that carbohydrate oligosaccharides in these agricultural residues have activity to stimulate the immune system and prevent chronic diseases such as cancer.  Commercialization of these new carbohydrates will add value to U.S. specialty crops and benefit consumer health.
                                Technical Abstract: Background
   Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body.  In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets including T-helper/inducer cell, Tcytotoxic/suppressor cell, B-cell, and Natural Killer (NK)-cells, and the induction of NK-cell
activity against K562 chronic myeloid leukemia cells.  A carbohydrate composition analysis is performed to propose a structural mechanism of action of immune enhancement by MCP.

Methods
   Human blood samples were collected and incubated with increasing concentrations of MCP along with appropriate positive controls.  The blood samples were mixed with antibody, incubated, lyzed and run on a Coulter Elite flow cytometer using a 3-color protocol.  The percentage of activated T-helper/inducer cells, T-cytotoxic/suppressor cells, B-cells and NK-cells and the percentage increase over untreated control were calculated and plotted against MCP concentrations.
   To test the functionality of the activated NK-cells, normal lymphocytes were isolated, washed, plated and treated with increasing concentrations (0-800 ug/ml) of MCP and positive
controls.  Log-phase PKH26-labeled K562 leukemic cells (0.2 x 106) were added to the normal lymphocytes and incubated for another four hours for inducing cell death.  The cell mixture was permeabilized, washed, and stained with human specific FITC-labeled active form of caspase 3 antibody.  The stained cells were washed, and analyzed by a two color flow cytometry protocol with FL1 and FL2 measuring PKH26 and FITC, respectively.  The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells.
   Statistical analysis: Mean and standard deviation values were calculated and graphs prepared by Microsoft Excel.  Kruskal-Wallis one-way Analysis of Variance (ANOVA) was used to analyze the data and p values were estimated by the GraphPad Prism software.
   Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD).  Weight average molar mass and intrinsic viscosity analysis was performed by high-performance size-exclusion
chromatography (HPSEC) with multiple detectors (multi-angle laser light scattering, refractive index and differential pressure viscometer).

Results
   Results show that MCP does not have a significant effect on T-helper/inducer cell activation as compared to positive control. However, results show that MCP activated Tcytotoxic/suppressor cells at low levels and in a dose-dependent manner, B-cell in a
significant dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-Activated NK-cells demonstrated functional activity in inducing cancer cell death.  The MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends.  Unsaturated oligogalacturonic acids have been reported with immunomodulatory activity.

Conclusions
   The data demonstrates that MCP is a substance with strong immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture.  Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP.  Additional in vivo studies to better understand the applications of MCP as an immune enhancer are warranted.


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