The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.ImagesFigure 1Figure 2
SUMMARY1. A study has been made of the effects of changing [Ca]. and [Mg]. on the binomial statistic parameters p and n which control the average quantal content (mi) of the synaptic potential due to acetylcholine release. 4. The facilitated increase in m9 during a short train was due to an increase in n, whereas the post-tetanic increase in mi during a tetanus was due to an increase in p. These results are considered in terms of the role of Ca ions in facilitation and post-tetanic potentiation.
Study Design
In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs.
Objectives
To examine the biologic effect of “cocktail” therapeutic gene transfer to human IVD cells in three dimensional cultures.
Summary of Background Data
. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted.
Materials and Methods
Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immuno-sorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads.
Results
Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control.
Conclusion
Combination or “cocktail” gene therapy offers a promising mechanism for maximizing matrix synthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.
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