AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells.

BACKGROUND AND PURPOSE: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs). EXPERIMENTAL APPROACH: MDDCs derived from healthy and hepatitis C virus (HCV)-infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T-lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase-based reporter methods, and Toll-like receptor (TLR)-blocking antibodies were employed to analyse TLR activity. KEY RESULTS: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA-DR, chemokines and chemokine receptors, interleukin (IL)-12p70 and IL-10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF-kappaB and phosphorylation of p38 mitogen-activated protein kinase. AM3 promoted NF-kappaB activation in a TLR-4-dependent manner, and blocking TLR-4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation-associated markers in MDDCs from HCV-infected patients and increased the proliferation of T lymphocytes induced by these MDDCs. CONCLUSIONS AND IMPLICATIONS: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation.

Phenolic components and antioxidant activity of Fernblock, an aqueous extract of the aerial parts of the fern Polypodium leucotomos.

Fernblock, an aqueous extract of the aerial parts of the fern Polypodium leucotomos, used as raw material for topical and oral photoprotective formulations, was fractioned by HPLC and the main components with antioxidant capability were identified by means of UV spectra, electrochemical detection, and MSn. Phenolic compounds were identified as 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, caffeic acid, 4-hydroxycinnamic acid, 4-hydroxycinnamoyl-quinic acid, ferulic acid, and five chlorogenic acid isomers. Total ferric antioxidant capacity (FRAP) of HPLC eluted fractions was measured. The results suggest that the herein identified compounds support, at least partially, the antioxidant and radical scavenging capacities of Fernblock.

Polypodium leucotomos extract: antioxidant activity and disposition.

The extract of the fern Polypodium leucotomos (PL, Fernblock) is an oral photoprotectant with strong antioxidative properties. Recent studies to determine its chemical composition have shown 4-hydroxycinnamic acid (p-coumaric), 3 methoxy-4-hydroxycinnamic acid (ferulic), 3,4-dihydroxycinnamic acid (caffeic), 3-methoxy-4-hydroxybenzoic acid (vanillic) and 3-caffeoilquinic acid (chlorogenic) to be among its major phenolic components. No conclusive data are available, however, on the H2O2-scavenging capacity of these compounds, or on their absorption and metabolism following their oral intake. In the present work, their antioxidative capacity was assessed by the luminol/H2O2 assay, their absorption studied using Caco-2 cells to resemble the intestinal barrier, and their metabolism investigated using cultured primary rat hepatocytes. The antioxidant capacity of PL components increased in a concentration-dependent manner, with ferulic and caffeic acids the most powerful antioxidants. The apparent permeability results correspond to a human post-oral administration absorption of 70-100% for all tested substances. Coumaric, ferulic and vanillic acids were metabolized by CYP450-dependent mono-oxygenases and partially conjugated to glucuronic acid and sulfate. These phenolic compounds may contribute to the health benefits afforded by this oral photoprotectant.

Inmunoferon, an immunomodulator of natural origin, does not affect the rat liver cytochrome P-450 and phase II conjugation enzymes.

Inmunoferon is a glycoconjugate of natural origin with immunomodulatory properties. It has recently been shown to regulate TNF-alpha expression induced by lipopolysaccharide (LPS) challenge through a hypothalamo-pituitary-adrenal (HPA)-dependent mechanism. Inmunoferon is orally administered to immunocompromised patients as an adjuvant during immune therapy such as vaccination or infectious diseases treatment. Due to its mainly adjuvant nature, it is necessary to determine if coadministration of Inmunoferon affects the activity of other drugs. In this study we analyzed the possible modification of the hepatic drug biotransformation system by using Inmunoferon in a rat model, which may result in changes in the biological activity of other drugs administered simultaneously. Inmunoferon-treated animals showed no differences to control littermates in antipyrine metabolism. No differences were found in either cytochrome P-450 and b5 levels or cytochrome P-450-dependent activities and phase II conjugation enzymes in lysates from Inmunoferon-treated rat hepatic cells. The same treatment reduced levels of serum TNF-alpha in LPS-challenged animals. In summary, Inmunoferon is unable to affect the hepatic bioconjugation system during administration and thus seems unlikely to interact with, or modify the effect of, coadministered drugs.

Identification and characterization of the peptidic component of the immunomodulatory glycoconjugate Immunoferon.

Inmunoferon is a glycoconjugate of natural origin, formed by the noncovalent association of a protein from Ricinus communis and a polysacharidic moiety, and endowed with immunomodulatory as well as pharmacological activities. This study investigated the nature of polypeptidic component of Inmunoferon. Through biochemical procedures and comparison with protein databases, the isolated protein was identified as the processed form of the seed of Ricinus communis 2S storage polypeptide, which has been termed RicC3. Further analysis of the isolated protein has revealed that it is composed of two different subunits, alpha and beta, which form an heterodimer of high stability and resistance to denaturation, acidic pH and proteolytic cleavage. These findings confirm the excellent properties of the product after oral administration and provide additional support for the pharmacological activities of Inmunoferon.

Inmunoferon, a glycoconjugate of natural origin, regulates the liver response to inflammation and inhibits TNF-alpha production by an HPA axis-dependent mechanism.

In this report, we have examined the mechanism of action of Inmunoferon, a patented glycoconjugate (GC) of natural origin with immunomodulatory properties, in the regulation of TNF-alpha expression induced by LPS challenge in a rodent model. GC was found to be dependent on a normal HPA response to exert its regulatory effect on TNF-alpha expression. Adrenalectomized mice were unresponsive to the drug in terms of TNF-alpha levels after LPS challenge, whereas control mice showed lower serum TNF-alpha levels when treated with GC than vehicle-treated animals. GC treatment also induced the expression of acute phase proteins but it did not alter the normal metabolism or viability of hepatic cells. These data suggested the employment of GC as a novel adjuvant during antibacterial treatment without disadvantages such as side effects in the metabolism of the liver, thus, acting as an enhancer of the host response against infection.

Immunoferon, a glycoconjugate of natural origin, inhibits LPS-induced TNF-alpha production and inflammatory responses.

We have analyzed the effect of a patented glycoconjugate (GC) of natural origin, Inmunoferon, in the development of the response to endotoxemia induced by administration of LPS in rodents. We have observed that oral treatment with the drug reduced the levels of serum TNF-alpha induced by an intravenous pulse of LPS. The serum of pretreated mice blocked TNF-alpha production by peritoneal macrophages. The drug increased the levels of TNF-alpha regulators such as IL-10 and corticosteroids, whereas it inhibited TNF-alpha-dependent IL-6 production. Further TNF-alpha-dependent responses, such as cell extravasation, was decreased in treated mice. According to these results, Inmunoferon is postulated as an inhibitor of the systemic response to LPS. Correlation of the observations made in mice with a rat model suggests the efficacy of this product in reducing TNF-alpha production in a species-independent fashion and opens the possibility of its trial as an adjuvant of antibiotics in treatment against gram-negative bacterial infection.

A new method for radioiodination of polysaccharides and its use in biodistribution studies of an immunomodulating glycoconjugate (Immunoferon).

A new method for the labeling of polysaccharides by using tyrosine derivatives after CNBr activation is presented, along with its use for pharmacokinetic studies of an orally active immunomodulating noncovalent glycoconjugate, which was administered labeled either in the polysaccharide or the protein moiety. The relationship between the obtained results and the mechanism of action are also discussed.