Evaluation of effects on immune surveillance, anti-bacterial, and inflammatory behavior of the PMN cell:

The PMN cell is a highly active and migratory cell type. The migratory behavior of this
cell type is divided into at least two types: a) random migration and b) directed migration.
Random migration is part of normal immune surveillance, whereas directed migration
happens towards specific chemoattractants.

We have established a migration test where both types of migration are tested in parallel.
Furthermore, the directed migration is tested towards three distinctly different chemotactic
compounds:

i) bacterial peptide f-Met-Leu-Phe;
ii) the inflammatory cytokine Interleukin-8 (IL-8); and
iii) the inflammatory mediator Leukotriene B4 (LTB4).

Previously, we have found interesting evidence during evaluation of several natural
products that some products may specifically reduce directed PMN migration towards the
inflammatory mediators IL-8 and/or LTB4 while allowing PMN migration towards
bacterial peptide as part of the normal anti-bacterial immune defense. We hoped to see a
similar effect with Zrii.

Freshly purified PMN cells were set up cultures in double-chamber migration plates, where
the bottom chamber mimics tissue, and the top chamber mimics the blood stream. Cells
were plated in the top chambers with and without serial dilutions of test product, and
different chemo-attractants were present in the bottom chambers. Control wells included
cells un-exposed to test products and without chemo-attractant in the bottom wells, and
allowed evaluation of baseline random migration.

The assay was performed three times using freshly isolated cells from different healthy
human donors. Within each test, controls were performed in quadruplicate, and tests in
duplicate.

The four graphs below shows the increase in migratory behavior under the four different
test conditions.

In these graphs, the value “1.00” is the baseline, i.e. the level of migratory activity without
Zrii-treatment of the cells. Values higher than “1.00”indicates that Zrii induced an increase
in migratory behavior. Values lower than “1.00” indicates that Zrii reduced the migration.
Zrii-treatment of PMN cells resulted in a dose-dependent increase in random and f-MLP
directed migration.

Thus, the data showed that Zrii supported two aspects of PMN cell
behavior in vitro, associated with a healthy immune defense in vivo.

As can be seen from the graphs on the next page, Zrii did not have a clear effect on IL-8
directed migration. In contrast, Zrii had a strong, pronounced anti-inflammatory effect
on LTB4 directed migration.

The trend to an anti-inflammatory effect, seen at the lowest dose of Zrii, led to testing
across an extremely wide dose range of Zrii. Data from this experiment is shown in a
separate graph below. The dashed line shows the baseline level of PMN migration when
no test product was added.

Conclusion
Zrii provided a protective effect in both the testing of apoptosis and mitochondrial
function, when PMN cells were used. PBMC showed a similar trend, but much less
pronounced.

Zrii supported two important aspects of PMN immune function, namely random migration
and migration towards the bacterial peptide f-MLP.

Zrii strongly inhibited PMN migration towards the inflammatory mediator LTB4.

Thus, Zrii supported cellular viability and energy formation, as well as key aspects of the
innate (immediate) anti-bacterial immune response. However, the antioxidant capacity and
the anti-inflammatory properties of Zrii would indicate that Zrii may boost the immune
defense mechanisms while protecting the body’s own cells from the resulting oxidative
stress.

Future research
Zrii’s anti-inflammatory effects are very promising. The low dose required to inhibit PMN
cell migration has not been explored to its fullest yet – even at a dose of 10 femtoliter per
liter culture showed a strong effect, and we have not yet been able to identify the lowest
active dose.