Predictive graphical model, network-based medical tool for the prognosis of chronic hepatitis C patients treated with peg-interferon plus ribavirin.

BACKGROUND: There are few model networks to predict treatment outcome in viral hepatitis. AIM: To develop an easy bioinformatics platform based on algorithm decisions (Bayesian network) for a more efficient prediction of treatment response. METHODS: Totally 385 consecutive chronic hepatitis C (CHC) treated patients were included. More than 40 variables were analysed. Data from 308 patients were used to build the variable model network using DLIFE platform based on predictive graphical models. The prediction accuracy of the bioinformatics network was compared with the true data collected in a retrospective study. The model was then validated twice with external data from CHC patients treated in other hospitals. RESULTS: The accuracy of this bioinformatics network for treatment response in our 308 patients was 83.3%, which is higher than the accuracy obtained by physicians on the basis of study of clinical data and their own experience (50-65%). The receiver operator characteristic curve areas after validation with another cohort of patients were: 0.91 for sustained virological response, one for nonresponse, and 0.81 for relapse. DLIFE offered a diagnostic accuracy of 81.3%, which is a clear improvement compared with unassisted prognosis (50-65%). CONCLUSIONS: This bioinformatics platform (DLIFE) accurately predicts the outcome of CHC combination therapy, improving treatment decisions and reducing costs. This bioinformatics platform allows integrating widespread data sources and permits predicting the clinical outcome of a particular patient using a general predictive graphical model.

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.

Molecular basis for the regenerative properties of a secretion of the mollusk Cryptomphalus aspersa.

A screen for natural products bearing pharmacological properties has yielded a secretion of the mollusk Cryptomphalus aspersa (SCA), which possesses skin-regenerative properties. In this report, we outline some of the cellular and molecular effects underlying this observation. First, we found that SCA contained antioxidant SOD and GST activities. In addition, SCA stimulated fibroblast proliferation and rearrangement of the actin cytoskeleton. Additional mechanisms involved in the regenerative effect of SCA included the stimulation of extracellular matrix assembly and the regulation of metalloproteinase activities. Together, these effects provide an array of molecular mechanisms underlying SCA-induced cellular regeneration and postulate its use in regeneration of wounded tissue.

Development of phosphorylated glucomannan-coated chitosan nanoparticles as nanocarriers for protein delivery.

The aim of the present work was to develop a new nanoparticle carrier, adapted for the oral administration of proteins and their delivery to the immune system. Chitosan and phosphorylated glucomannan were chosen as major constituents of the nanoparticles. Chitosan nanoparticles were formed by ionic gelation and then coated with glucomannan. Two different protocols were adopted for the formation of the glucomannan coating: protocol I, in which chitosan nanoparticles were isolated before their coating; protocol II, in which chitosan nanoparticles were not isolated, but coated with glucomannan in the presence of free chitosan. The results showed that, under the selected formulation conditions, the sizes of the nanoparticles ranged between 170 and 300 nm and their zeta potential values were inverted from positive to negative by the glucomannan coating. The nanoparticles prepared by the two protocols could be freeze-dried, in the presence or absence of cryoprotective agents, preserving their original characteristics. The results of the stability study evidenced the positive role of the glucomannan coating in preventing the aggregation of the nanoparticles in buffered media. Finally, the association of the inmunomodulatory protein complex P1 to the chitosan-glucomannan nanoparticles was investigated. The results showed that the association was not dependent on the chitosan: sodium tripoliphosphate ratio, but it was significantly affected by the presence of sodium phosphate in the protein structure.

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 inhibits trans-urocanic acid photoisomerization and photodecomposition.

In this report, we demonstrate a possible molecular mechanism by which a hydrophilic extract of the leaves of the fern Polypodium leucotomos (Fernblock, PL) blocks ultraviolet (UV)-induced skin photodamage. The extract inhibits UVA and UVB light induced photoisomerization of trans-urocanic acid (t-UCA), a common photoreceptor located in the stratum corneum, and also blocks its photodecomposition in the presence of oxidizing reagents such as H2O2, and titanium dioxide (TiO2). PL protects in vitro human fibroblasts from UV-induced death as well. These results suggest the potential of employing the PL extract as a component of sunscreen moistures in order to prevent photodecomposition of t-UCA, to inhibit UV-induced deleterious effects of TiO2 and to protect skin cells and endogenous molecules directly involved in skin immunosurveillance.

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.

AM218, a new polyanionic polysaccharide, induces radioprotection in mice when administered shortly before irradiation.

We have shown that the polyanionic polysaccharide AM218 improves the survival rate of the potentially lethally irradiated mice. This radioprotective effect was highly dependent on the administration schedule, the most efficient protocol being that in which the drug was given shortly before irradiation. The haematopoietic implications in the pharmacological action of AM218 were confirmed by the improved recovery in the three peripheral blood lineages observed in the AM218-treated mice. However, because of a marked increase observed in the number of white blood cells during the period of highest mortality of the control irradiated mice, effects on the neutrophil lineage may account for the effects mediated by AM218 in the irradiated mice. Both in vitro and in vivo treatment with AM218 before irradiation improved the survival rate of CFU-GM progenitors, while no effects were observed on the CFU-S pool. This led us to postulate that the improved survival rate of the committed progenitors, at least the granulocyte-macrophage progenitors, accounts for the radioprotective capacity of AM218.

Prevention of in vitro neutrophil-endothelial attachment through shedding of L-selectin by nonsteroidal antiinflammatory drugs.

The activation of the endothelial cells by extravascular stimuli is the key event in the extravasation of circulating leukocytes to target tissues. L-selectin, a member of the selectin family, is constitutively expressed by white cells, and is the molecule involved in the initial binding of leukocytes to activated endothelium. After activation, leukocytes rapidly release L-selectin from the cell surface, suggesting that the functional activity of this molecule is controlled in large part by its appearance and disappearance from cell surface. We have studied in a neutrophil-activated endothelial cell binding assay, the effect of different antiinflammatory drugs (steroidal and nonsteroidal) in the L-selectin-mediated interaction of neutrophils with activated endothelial cells. Some nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin, diclofenac, ketoprofen, and aspirin, but not steroids, strongly inhibited the neutrophil-endothelial cell attachment. Furthermore, we also investigated the underlying mechanism of this functional effect. The expression of L-selectin on the neutrophil surface rapidly decreased in the presence of different NSAIDs, in a dose- and time-dependent manner, whereas no changes in the expression of other adhesion molecules such as CD11a, CD11b, CD31, or ICAM-3 (CD50) were observed. Interestingly, studies in vivo on healthy volunteers treated with physiological doses of indomethacin showed a significant decrease of L-selectin neutrophil expression. Only diclofenac induced an upregulation of CD11b expression, suggesting an activating effect on neutrophils. No enzyme release was observed upon treatment of neutrophils with different NSAIDs, indicating a lack of degranulatory activity of NSAIDs, with the exception of diclofenac. The downregulation of L-selectin expression was due to the rapid cleavage and shedding of the membrane L-selectin, as determined by both immunoprecipitation from 125I-labeled neutrophils, and quantitative estimation in cell-free supernatants. These results suggest that NSAIDs exert a specific action on adhesion receptor expression in neutrophils, which might account, at least in part, for the antiinflammatory activities of NSAIDs.

AM5, a protein-associated polysaccharide, stimulates hematopoiesis and modulates the expression of endogenous hematopoietic growth factors in murine long-term bone marrow cultures.

We have investigated the mechanism behind the hematopoietic stimulation mediated by AM5, a protein-associated polysaccharide that stimulates in vivo the murine hematopoietic system. A dose-dependent increase in hematopoietic progenitors was observed in long-term bone marrow cultures (LTBMCs) treated in vitro with AM5. The stimulatory effect was more marked in colony-forming units-granulocyte-macrophage (CFU-GM) than in CFU-spleen (CFU-S) progenitors and also more significant in the supernatant than in the adherent layer. This stimulatory effect was reversible, and continuous stimulation with high doses of AM5 was conductive to a progressive exhaustion of the culture. The analysis of the CFU-GM stimulating activity present in the supernatant of AM5-treated cultures revealed a dose-dependent induction of granulocyte-macrophage colony-stimulating activity (GM-CSA), in contrast with control cultures in which no CSA was detected. Northern blots of LTBMC-adherent layers obtained after in vitro treatment with AM5 revealed a significant mRNA expression for interleukin 1 alpha (IL-1 alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (M-CSF) and granulocyte CSF (G-CSF), in contrast with adherent layers from untreated cultures which only expressed, in detectable levels, M-CSF and stem cell factor (SCF). The SCF expression was down-modulated in AM5-treated cultures. Our results strongly suggest that the hematopoietic stimulation induced by AM5 is mediated by the modulated expression of endogenous hematopoietic growth factors.

Induction of tumor necrosis factor alpha production by human hepatocytes in chronic viral hepatitis.

Tumor necrosis factor alpha (TNF-alpha) is a multifunctional cytokine that has an important role in the pathogenesis of inflammation, cachexia, and septic shock. Although TNF-alpha is mainly produced by macrophages, there is evidence regarding TNF-alpha production by cells that are not derived from bone marrow. TNF-alpha production by normal and inflamed human liver was assessed at both mRNA and protein levels. Using a wide panel of novel anti-TNF-alpha monoclonal antibodies and a specific polyclonal antiserum, TNF-alpha immunoreactivity was found in hepatocytes from patients chronically infected with either hepatitis B virus (HBV) or hepatitis C virus. Minimal TNF-alpha immunoreactivity was detected in the mononuclear cell infiltrate and Kupffer cells. In situ hybridization experiments using a TNF-alpha RNA probe showed a significant expression of TNF-alpha mRNA in hepatocytes, Kupffer cells, and some infiltrating mononuclear cells. By contrast, TNF-alpha was detected at low levels in liver biopsies from normal individuals or patients with alcoholic liver disease and low expression of TNF-alpha mRNA was observed in these specimens. Transfection of HepG2 hepatoblastoma cells with either HBV genome or HBV X gene resulted in induction of TNF-alpha expression. Our results demonstrate that viral infection induces, both in vivo and in vitro, TNF-alpha production in hepatocytes, and indicate that the HBV X protein may regulate the expression of this cytokine. These findings suggest that TNF-alpha may have an important role in human liver diseases induced by viruses.

Signaling through the LFA-1 leucocyte integrin actively regulates intercellular adhesion and tumor necrosis factor-alpha production in natural killer cells.

The LFA-1 leucocyte integrin is known to participate in natural killer (NK) cytolytic activity, mediating effector target interactions. The possibility that LFA-1 may also play an active regulatory role in NK cells has been explored. To this end, we have employed a monoclonal antibody (HP1N) raised against recombinant interleukin-2 (rIL-2)-activated NK cells, which recognizes the alpha chain of the LFA-1 heterodimer (CD11a). In contrast to other anti-CD11a mAb the HP1N and its F(ab')2 fragment did not affect NK cell-mediated cytotoxicity and triggered a strong homotypic adhesion of NK cells and other LFA-1+ cells. Cellular aggregation was inhibited by anti-CD18 mAb, anti-ICAM-1 mAb, and other anti-CD11a mAb. Remarkably, the HP1N mAb was also shown to induce tumor necrosis factor-alpha (TNF-alpha) production from NK cells upon costimulation with anti-CD16 mAb. Such an effect appeared to be independent from homotypic adhesion since it took place in Mg(2+)-free medium, where NK cell aggregation was inhibited. Moreover, incubation with the HP1N mAb triggered a Ca2+ influx into the cytosol; this effect was clearly observed upon cross-linking of cell bound HP1N and was also substantiated with other anti LFA-1 (CD11a and CD18) mAb. Taken together these results indicate that the LFA-1 molecule is capable of transducing signals in NK cells, which regulate the intercellular interaction with its ligand, and enhance the activation via Fc gamma receptor type III.

Tumor necrosis factor-alpha production induced in T lymphocytes through the AIM/CD69 activation pathway.

Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation, which has been also involved in lymphocyte proliferation. Although AIM is absent from peripheral blood resting lymphocytes, it is expressed by in vivo activated lymphocytes infiltrating sites of chronic inflammation in several pathologies, as well as by lymphocytes after in vitro activation with different stimuli. We have investigated the possibility that tumor necrosis factor-alpha (TNF-alpha) gene expression and protein secretion could be induced in peripheral blood T cells through the AIM/CD69 molecule. Anti-AIM monoclonal antibodies (mAb) were able to induce TNF-alpha secretion in T cells when protein kinase C (PKC) was simultaneously activated by treatment with phorbol esters. TNF-alpha secretion was detected at 24 h and peaked at day 3 upon T lymphocyte activation with anti-AIM mAb. Immunoprecipitation studies with an anti-TNF-alpha mAb from surface iodinated T cells activated through AIM, demonstrated that TNF-alpha first appeared as a cell surface molecular form of 26 kDa, which is subsequently released to the extracellular medium as the 17-kDa molecular form of TNF-alpha. AIM stimulation dramatically increased TNF-alpha mRNA levels, and this mRNA induction and subsequent TNF-alpha secretion were virtually abrogated by the immunosuppressive drug cyclosporin A. Taken together these results indicate that AIM constitutes a novel molecular pathway in T lymphocytes for induction of TNF-alpha, and suggest a relevant pathologic role for AIM+ lymphocytes located at sites of tissue injury in the pathogenesis of different chronic inflammatory diseases.

Significant anti-HIV activity of new modified polyanionic polymers in vitro.

The anti-HIV activities of two new polyanionic polymers (AM 242 and AM 612) were investigated in cell culture-based and biochemical antiviral assays. These compounds inhibited the reverse transcriptases from HIV-1 and HIV-2, using enzyme purified from virions and either a ribosomal RNA or gapped duplex DNA as the template. With the ribosomal RNA template, AM 242 and AM 612 had ID50 values of 1.1 and 0.10 micrograms/ml against the HIV-1 reverse transcriptase. In vitro cell based assays determined that both compounds significantly inhibited both the cytopathic effects associated with HIV-1 infection and the replication of virus in infected cells. AM 242 had an IC50 of approximately 1.0 micrograms/ml, while that of AM 612 was 0.19 micrograms/ml. These two active polyanionic polymers were effective in inhibiting the growth of a panel of HIV-1 isolates and were also active against HIV-2. Although the compounds were toxic at high concentration, they had antiviral activity over a wide range of nontoxic concentrations, yielding a high selectivity index. AM 612 was 100% protective for CEM cells from 320 ng/ml to 1 microgram/ml. Both compounds caused a significant increase in cellular proliferation as determined by the concentration-dependent increase in incorporation of radioactive precursors into cellular macromolecules.

Down-regulation by tumor necrosis factor-alpha of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism.

Adhesion of human neutrophils to endothelial cells is a crucial step during migration to the extravascular sites of inflammation. A large number of molecules, including the CD44 and LAM-1 antigens, have been described to participate in this process. We have investigated the regulation by human recombinant tumor necrosis factor-alpha (TNF-alpha) of human neutrophil plasma membrane expression of both CD44 and LAM-1 adhesion molecules, as well as that of CD43 sialophorin, which has been involved in adhesion and activation of leukocytes. The expression of these three antigens was down-regulated in neutrophils upon TNF-alpha treatment, as determined by immunofluorescence and immunoprecipitation experiments. However, the expression of other cell surface molecules, such as CD45 or CD11b, was up-regulated. Similar regulatory effects were also observed upon neutrophil treatment with other activating agents such as the chemoattractant peptide formyl-Met-Leu-Phe, the calcium ionophore A23187, or the phorbol ester phorbol 12-myristate 13-acetate. Protease inhibitors virtually abrogated the TNF-alpha-induced down-regulation of CD43 and CD44 expression, but not that of LAM-1, suggesting the involvement of a protease activity in this process. These results underline the role of TNF-alpha on the differential regulation of cell surface expression of neutrophil adhesion molecules, thus implying modifications in the neutrophil adhesive properties.