In Vitro Study of a polysaccharide extract in human lymphocytes
Cell isolation and culture: Peripheral blood mononuclear cells (PBMC) from healthy donors were
isolated using standard Ficoll-Hypaque density gradient centrifugation and resuspended in RPMI1640 culture medium, supplemented with 10% fetal bovine serum. Cells were incubated for different
periods (1-4 days) with various concentrations of the polysaccharide extract (100 ng/ml - 100 µg/ml)
alone, with interleukin (IL)-2 (20-1000 IU/ml) alone (as for a positive control), or with both factors
(polysaccharide/IL-2) and were subsequently tested in standard proliferation and cytotoxicity assays.
Proliferation assay: PBMC from 5 different healthy donors were seeded in 96-well plates (2 x 105
cells/well) and incubated in the presence of polysaccharide, IL-2 or polysaccharide/IL-2 for 3 and 5
days. During the last 18 hours, cultures were pulsed with radioactive thymidine ( 3[H]-TdR; 1
µCi/well), which is incorporated into the DNA of proliferating cells. Cells were further harvested on
glass fiber filters, using a semi-automated cell harvester and upon addition of scintillation fluid,
radioactivity was counted in a β-counter. The polysaccharide extract was tested at escalating
dilutions, ranging from 100 ng/ml - 100 µg/ml. The results show that upon a 3-day incubation, the
substance can significantly induce lymphocyte proliferative responses (1.3-2.8 fold increase,
compared to the proliferation of the negative controls), at dose level ranging from 0.5 – 20 µg/ml of
polysaccharide. In one of the donors, incubation of PBMC with 100 µg/ml of polysaccharide for 5
days, resulted in a more pronounced increase of proliferation, which was by 7.26-fold higher than
that of the respective negative controls. Finally, higher proliferation of lymphocytes was noticed
when the polysaccharide was tested in synergy with suboptimal concentrations of IL-2 (20 IU/ml).
The relevant increase of proliferation, after 3 days of incubation with optimal concentrations of the
polysaccharide (0.5-20 µg/ml), was by 2.5-3.9 fold higher compared to the respective negative
controls.
Cytotoxicity assay: Tumor targets (T) from the human cell lines K562 (chronic myeloid leukemia)
and Daudi (Burkitt lymphoma) were incubated with radioactive chromium (Na251CrO4) for 90 min at
37o C. Effector cells (E), deriving from PBMC of 6 different healthy donors, were preincubated for
1-3 days with polysaccharide, IL-2 or polysaccharide/IL-2, plated in 96-well plates (1-2 x 105
cells/well) and incubated with the 2 labeled targets at an E:T ratio of 40:1. As for controls, target
cells were incubated in plain medium and with 3N HCl, for estimations of spontaneous and maximal
isotope release, respectively. After the end of the incubation, 100 µl of supernatant were removed
from each well for isotope counting in a γ-counter. Percentage-specific 51Cr release was calculated
according to the formula: counts per minute [cpm] sample - cpm spontaneous x 100 cpm maximum cpm spontaneous. In accordance to the proliferation assays, the polysaccharide extract was tested in a
wide range of concentrations, 100 ng/ml - 100 µg/ml. The substance was shown to enhance the
cytotoxic activity of natural killer (NK) cells against the NK-sensitive K562 and the NKresistant
Daudi tumor cell lines (by 1.5-2.5 fold, in both cases) at doses ranging from 1-10 µg/ml. The
induction of cytotoxicity was further augmented when the polysaccharide extract was tested along
with low doses of IL-2 (20 IU/ml). In the latter experiments, an overall enhancement of cytotoxicity
by 2.1-4.3 fold was observed. Finally, in kinetic studies performed in parallel, we noticed that the
stimulating effect of the polysaccharide on human PBMC was more pronounced upon short time
incubation, with an optimal activation time of 1-2 days, whereas prolonged PBMC exposure to the
substance did not seem to further enhance the lytic ability of effector cells. Specifically, at a
polysaccharide dose of 10 µg/ml, the percentage of cytotoxicity upon incubation of PBMC for 1, 2, 3
and 4 days was 38.7 %, 30.5 % 24 % and 11.3 %, respectively (% cytotoxicity of negative control
6.9 %). Synergistically with IL-2, the optimal incubation time was determined at 2 days, and the
respective percentages of cytotoxicity for 1, 2, 3 and 4 days were 40.7 %, 60.2 %, 41.6 % and 30 %,
respectively (% cytotoxicity of negative control 13.2 %).
The aforementioned in vitro results are only indicative, performed on PBMC samples deriving
from a limited number of individuals. A series of more detailed experimentations is required to
investigate the mode of action of the polysaccharide extract and to isolate and identify the
active compound/compounds present in it. Moreover, in order to establish the
immunomodulating effect of the polysaccharide extract, the design of in vivo experiments in
animal models (preferably in mice) will be required.
Constantinos Vorgias and Rania Tsitsiloni
Athens University