Natural killer cell activation and modulation of chemokine receptor profile in vitro by an extract from the cyanophyta Aphanizomenon flos-aquae.

The present research was designed to study the effects of an extract from the edible cyanophyta Aphanizomenon flos-aquae on human natural killer (NK) cells. We have previously shown, using a double-blind randomized placebo-controlled crossover design, that ingestion of 1.5 g of dried whole A. flos-aquae resulted in a transient reduction in peripheral blood NK cells in 21 healthy human volunteers, suggesting increased NK cell homing into tissue. We have now identified an extract from A. flos-aquae (AFAe) that directly activates NK cells in vitro and modulates the chemokine receptor profile. NK cell activation was evaluated by expression of CD25 and CD69 on CD3-CD56+ cells after 18 hours. Changes in CXCR3 and CXCR4 chemokine receptor expression after 5-60 minutes were evaluated by immunostaining and flow cytometry. AFAe induced the expression of CD69 on CD3-CD56+ NK cells, induced CD25 expression on 25% of these cells, and acted in synergy with interleukin 2. NK cells enriched by RosetteSep (StemCell Technologies Inc., Vancouver, BC, Canada) were not activated by AFAe, indicating that the NK activation was dependent on other cells such as monocytes. The low-molecular-weight fraction <5,000 of AFAe was responsible for the most robust NK cell activation, suggesting novel compounds different from previously reported macrophage-activating large polysaccharides.

Immunomodulation by SanPharma fungal metabolic products.

OBJECTIVE: In the present study, a series of fungal metabolite products from SanPharma (Dohren, Germany) were tested for effects on human peripheral blood leukocytes in vitro using standard immunologic methods. BACKGROUND: Therapeutic strategies used in German biological medicine often include treatment (oral, nasal, rectal, topical, or injection) with fungal or bacterial products, also known as "isopathic remedies," of which some are limited to metabolic products, whereas others include microbial cell lysates and cell wall fragments as well. The SanPharma products are based on metabolites, and do not contain microbial cell wall compounds. METHODS: Activation of natural killer (NK) cells was evaluated by cell surface immunostaining using CD3, CD56, CD69, and CD25 monoclonal antibodies. Production of interferon-gamma was evaluated by enzyme-linked immunoabsorbent assay (ELISA) on supernatants collected after 5 days' culture of peripheral blood mononuclear cells (PBMC). Direct mitogenic effect was assessed using the lipophilic membrane dye PHK26 (Sigma-Aldrich, St. Louis, MO) in a fluorescence-based proliferation assay, in which fluorescence intensity is reduced upon cell divisions. Cell viability upon exposure to fungal metabolites was assessed using propidium iodide staining and flow cytometry. RESULTS: All fungal metabolite products specifically induced the expression of the CD69 marker on human CD3-negative, CD56-positive NK cells, but not CD3-positive T cells, in vitro, as shown by the induction of the CD69 marker on up to 50% of NK cells after 18 hours' culture with metabolites. Only one of the five metabolite products, Roqueforti, induced cyclooxygenase-2 (COX-2), indicating that nuclear factor-kappaB (NFkappaB)-mediated signaling may not have been involved in the NK activation by the other four products. The Notatum product reduced baseline levels of COX-2, indicating an anti-inflammatory effect. No evidence of toxic or mitogenic effects was found. CONCLUSIONS: The fungal metabolite products from SanPharma specifically activate human NK cells in vitro.