Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

Immunomodulation by shark cartilage extracts

The immune system is composed of innate and adaptive mechanisms. Innate immune responses are significantly modulated by immunomodulatory factors that act through the induction of specific patterns of cytokine production in responding cells. Human leukocytes have been shown to respond to substance(s) present in acid extracts of commercial shark cartilage (SC). Shark cartilage is a food supplement taken by consumers as a prophylaxis and for the treatment of conditions ranging from arthritis to cancer. No reliable scientific evidence in the literature supports the alleged usefulness of shark cartilage supplements, but their use remains popular. Cartilage extracts exhibit immunomodulatory properties by inducing various inflammatory, Th1-type cytokines and potent chemokines in human peripheral blood leukocytes (HPBL) in vitro. The objectives of the study were to (1) to determine the nature of the active component(s), (2) to define the scope of cellular response to SC extract, and (3) to elucidate the molecular mechanisms underlying bioactivity. Results showed that there are at least two cytokine-inducing components which are acid stable. One anionic component has been identified as a small (14-21 kDa) glycoprotein with at least 40% carbohydrate content. Shark cartilage stimulated HPBL to produce cytokines resembling an inflammatory, Th1 polarized response. Leukocyte-specific responses consist of both initial cytokine responses to SC directly (i.e., TNF-α) and secondary responses such as the IFN-γ response by lymphocytes following initial SC stimulation. Response of RAW cells, a murine macrophage cell line, indicated that TNF-á could be induced in macrophages of another mammalian species in the absence of other cell types. The results suggest that the human monocyte/macrophage is most likely to be the initial responding cell to SC stimulation. Stimulation of cells appears to engage at least one ligand-receptor interaction with TLR 4, although the role of TLR 2 cannot be ruled out. Initial activation is likely followed by the activation of the JNK and p38 MAPK signal transduction pathways resulting in activation, release, and translocation of transcription factor nuclear factor κB (Nf-κB). This dissertation research study represents the first in-depth study into characterizing the bioactive component(s) of commercial shark cartilage responsible for its immunomodulating properties and defining cellular responses at the molecular level.

Antibacterial proteins and peptides in nurse shark ( Ginglymostoma cirratum) peripheral blood leukocytes

In many vertebrate and invertebrate species mediators of innate immunity include antimicrobial peptides (AMPs) such as peptide fragments of histones and other proteins with previously ascribed different functions. Shark AMPs have not been described and this research examines the antibacterial activity of nurse shark (Ginglymostoma cirratum) peripheral blood leukocyte lysates. Screening of lysates prepared by homogenizing unstimulated peripheral blood leukocytes identified muramidase (lysozyme-like) and non-muramidase antibacterial activity. Lysates were tested for lysozyme using the lysoplate assays, and antibacterial (AB) activity was assayed for by a microdilution growth assay that was developed using Planococcus citreus as the target bacterium. Fractionation of crude lysates by ion exchange and affinity chromatography was followed by a combination of SDS-PAGE with LC/MS-MS and/or N-terminal sequence analysis of low molecular weight protein bands (<20 kDa). This yielded several peptides with amino acid sequence similarity to lysozyme, ubiquitin, hemoglobin, human histones H2A, H2B and H4 and to antibacterial histone fragments of the catfish and the Asian toad. Not all peptide sequences corresponded to peptides potentially antibacterial. The correlation of a specific protein band in active lysate fractions was accomplished by employing the acid-urea gel overlay assays in which AB activity was seen as zones of growth inhibition on a lawn of P. citreus at a position corresponding to that of the putative AB protein band. This study is the first to describe putative AMPs in the shark and their potential role in innate immunity.^