STUDY TO ESTABLISH THE ACCEPTANCE RANGE FOR PEROXYL RADICALS SCAVENGER CAPACITY OF NATURAL SOD.

In the context of an emerging market of food supplements, the proven quality of the antioxidant products should be the main criteria for using them. The production process has to be carefully controlled and complementary tests are needed to demonstrate the correspondence between real and declared properties of final product. Using well characterized compounds with proven antioxidant activity in biological systems as reference brings a plus of rigorously to the testing protocol. The aim of this study was to determine the acceptance range for the antioxidant (peroxyl radicals scavenger) capacity of "Natural SOD" by using for comparison ascorbic acid (vitamin C). The established acceptance range complete our previous results concerning the antioxidant capacity of Natural SOD using validated ORAC method and creates premises for supplementary checking of the batches in the current production and improving the product quality.

Implant microparticles--a new concept for non-invasive cancer therapy.

Some progress in cancer research was possible in recent years mainly due to important advances in nanotechnology. However, clinical use of nanomaterials is still hindered by limitations. In search of better performance and control of inoculated materials, the efficiency and toxicity of SBBC implant particles was assessed. B16 tumoral cells (murine melanoma) were subjected to SBCC particles using in vitro and in vivo experimental models. In vitro experiments concerning the growth inhibition of tumoral cells using SBCC particles were performed by Flow Cytometry and by MTT Assay. In vivo experimental model (C57BL/6 mice) was used to complete this investigation: weight, viability and tumoral dimension were monitored. An anti-proliferative activity on B16 tumoral cells and an ability to produce apoptosis were observed. A reduction of tumoral volume and a 54% survival rate in the treated animals compared to the controls was obtained. Our preliminary results showed that the SBCC implants were effective against B16 melanoma cells, while there is no toxicity associated.

A highly purified vegetal fraction able to modulate HMGB1 and to attenuate septic shock in mice.

High-mobility group box protein 1 (HMGB1) is an intracellular protein that may be released actively from monocytes and macrophages or passively from necrotic or damaged cells. Its inhibition in animal experiments, even in the late phase of septic shock, significantly enhanced the survival rate of rodents. The aim of our study was to investigate the effect of a vegetal fraction isolated and highly purified from Helleborus purpurascens regarding the modulation of HMGB1 release either from tumor cells or human blood mononuclear cells. Our results showed that the vegetal fraction was able to down-regulate the release of HMGB1 from activated human blood mononuclear cells (PBMCs) and tumor cells. By combining the purified fraction with Cyclophosphamide the release of HMGB1 from tumor cells was strongly decreased. This synergism was not noticed when the ve getal product was associated with Doxorubicin. We also studied the effect of the purified fraction in mice with septic shock induced by cecal ligation and puncture (CLP) method. The tested vegetal product increased significantly the survival rate of animals compared to the mice not treated with it. Our data suggest that the purified vegetal fraction may modulate inflammation by down-regulating the HMGB1, which can also explain its efficacy in septic shock in mice.

The effects of some Curdlan derivatives on Dectin-1 expression and cytokine production in human peripheral blood mononuclear cells.

The cells of immune system such as monocytes and macrophages are in first line defence against dangerous signals. In the present paper the recognition of Dectin 1 receptors and the modulation of Interleukin-10 (IL-10) and Tumor Necrosis Factor-alpha (TNF-alpha) cytokine production by Curdlan and Curdlan derivatives in peripheral blood mononuclear cells (PBMCs) were studied. The effect of Curdlan or Curdlan derivatives on the expression of Dectin 1 receptors in PBMCs was revealed by flow-cytometry and the levels of IL-10 and TNFalpha were measured by ELISA kit in supernatants of PBMCs cultured in presence or absence of Curdlan, Curdlan derivatives and LPS. Our results suggested that Curdlan and Curdlan derivatives were able to increase the expression of Dectin-1 receptors on monocyte cells. The combined treatment of Curdlan/Curdlan derivatives and Pam3Cys produced an increase of CD14+ cells possessing Dectin-1 receptors. We demonstrated that Curdlan (at 20 microg unique dose) up-regulated TNF-alpha production and down-regulated IL-10 production in PBMCs. Conversely, Palm CM/SP-Curdlan (20 microg unique dose) was able to down-regulate TNF-alpha production and to up-regulate IL-10 production in PBMCs. For instance, Palm CM/SP-Curdlan determined a 5 times decrease of TNF-alpha production than Curdlan. Regarding the effect of Palm CM/SP-Curdlan on IL-10 production in PBMCs, we noticed that the level of IL-10 was about 4 times greater than Curdlan activity. We observed that a combined treatment of Curdlan/Curdlan derivatives and LPS induced about 5 times decrease in TNF-alpha production in PBMCs. IL-10 production induced by Palm CM/SP-Curdlan and LPS was about 6 times greater than the combined effect of Curdlan and LPS. The treatment of PBMCs with SP-Curdlan alone affected neither TNF-alpha production nor IL-10 production. Our results are in accordance with other studies demonstrating that Dectin-1 and TLR2/TLR6 signaling combine to enhance the responses triggered by each receptor and the signaling pathway induced by Dectin-1 could mediate the production of pro-inflammatory cytokines.

Curdlan derivatives able to enhance cytostatic drugs activity on tumor cells.

The chemotherapy success to kill cancer cells depends on its ability to stop cell division. The faster the cells are dividing, the more likely it is that chemotherapy will kill the cells, causing the tumor to shrink. Taking into account the severe side effects of chemotherapy, drugs producers also focus on natural products obtained either from medicinal plants, or from microorganisms. The complex polysaccharides named beta-glucans are active compounds with immune activity. beta-glucan polymers belong to a class of drugs with effects on the immune system, such as: anti-tumoral, anti-infectious, protection against fungi, bacteria and viruses infections. The correct selection of beta-glucans is essential to identify compounds with favorable clinical effects. The aim of this study was to investigate the capacity of six Curdlan (beta-glucan) derivatives to up-regulate the Doxorubicin, Actinomycin D and Cyclophophamide cytostatic drug activity on tumor cells (murine B16 melanoma and human HEp-2 laryngeal carcinoma cell lines). Our results demonstrated that Palm SP derivative, as well as SP and Palm CM/SP derivatives were able to potentiate Doxorubicin action or Actinomycin D effect on B16 tumor cells. SP derivative significantly enhanced cytostatic activity of Cyclophosphamide on B16 cells. All the investigated Curdlan derivatives (SP, Palm CM/SP, CM/SP, Palm CM, Palm SP and CM) were able to inhibit HEp-2 tumor cell growth, by up-regulating Doxorubicin and Actinomycin D cytostatic activity.

The modulation of reactive oxygen species production from human polymorphonuclear cells by curdlan derivatives as dectin-1 agonists/antagonists.

Reactive oxygen species (ROS) are well known to be cytotoxic and have been implicated in the etiology of a wide array of human diseases including diabetes, neurodegenerative diseases, cancer and also influence central cellular processes such as proliferation, apoptosis, senescence etc. If in these pathological or degenerative conditions characterized by free radicals excess, reactive species are not eliminated, they can maintain destructive processes, already initiated at different cellular levels. Understanding the role of ROS as key mediators in signaling cascades may provide various opportunities for pharmacological intervention. Toll-like receptors and C-type lectin receptor class V--Dectin-1, as members of Pattern Recognition Receptors play an essential role in innate immune response against bacteria and fungi respectively, contributing to pathogens recognition, phagocytosis, ROS production and induction of pro-inflammatory cytokines secretion. Using a high performance chemiluminometric method, we studied the action of six Curdlan derivatives on the ROS production and release by activated human polymorphonuclear cells (PMNs) isolated from the peripheral blood of healthy donors. Our results demonstrated that Curdlan derivatives containing sulfopropyl groups did not activate human PMNs to release ROS. These compounds blocked Dectin-1 and were able to inhibit co-operation between Dectin-1 and TLR-2. Curdlan derivatives containing palmithoyl, carboxi-methyl and sulfopropyl groups increased ROS release by human PMNs activated at TLR-2 level. Taking into account the fact that Dectin-1 can actively collaborate with TLR-2 to modulate the subsequent adaptive immune response, we can presume that Curdlan derivatives containing sulfopropyl group or palmithoyl/carboxi-methyl/sulfopropyl groups, as possible Dectin-1 antagonists/agonists, could influence TLR-2 signaling.

Recognition and modulation of Dectin-1 and TLR-2 receptors by curdlan derivatives and purified natural extracts.

Toll-like receptors (TLRs) and Dectin-1, as members of Pattern Recognition Receptors play an essential role in innate immune response against bacteria and fungi respectively, contributing to pathogens recognition, phagocytosis, etc. Dectin-1 and TLR-2/TLR-6 can interact for intracellular signal transduction. Dectin-1 is expressed at low levels on macrophages and at high levels on dendritic cells. Dectin-1 and TLRs are synergistic in mediating cytokines production, such as IL-12 and tumor necrosis factor alpha (TNF alpha). In the present paper we studied the expression of Dectin-1 (beta-Glucan Receptor C-type lectin receptor class V) and TLR-2 on human normal monocytes cells and also the role of different Curdlan derivatives and highly purified natural extracts, especially their capacity to recognize these receptors and their Dectin-1 agonist/antagonist properties. Our results demonstrated that Curdlan derivatives containing sulfopropyl or palmythoil/carboximethyl/sulfopropyl groups and natural extracts could be potent immunomodulators with many potential applications (possible antagonists of Dectin-1, blockers of Dectin-1 cooperation with TLR-2).

The effects of cold atmospheric plasma jets on B16 and COLO320 tumoral cells.

Cold atmospheric plasma treatment acts at the cellular level to remove diseased tissue without inflammation and damage, to suppress infections and to modulate the viability (apoptosis/necrosis) of tumoral cells. It is also known that, a major cause of anti-tumor chemotherapy failure is the development of multidrug resistance (MDR) of tumors. This study reveals the effect of high voltage pulsed, repetitive cold atmospheric plasma jets which are chemically activated with oxygen, on B16 tumoral cells (murine melanoma cell line) and COLO320DM multidrug resistant cells (human colon cancer cell line). The tests have been performed on human colon cancer cell line COLO320DM and murine melanoma cell line B16-F10. These cell lines have been treated with cold helium or helium-oxygen generated plasma jets and the consequent apoptosis has been analyzed by means of flow cytometric method. A treatment time-dependent apoptosis has been observed only in the case of 816-F10 cells interacting with helium-oxygen plasma and no apoptosis has been identified when the cells were treated only with helium plasma jets. These results indicate the need of oxygen for the chemical activation of plasma. The COLO320DM cells (that over-express the MDR efflux pumps) have been exposed to helium-oxygen plasmas only, or in a combination with vegetal extract MCS D161 as MDR efflux pumps inhibitor. For the secondly mentioned case the results have showed an increased apoptosis rate compared to the plasma treatment alone. The obtained data represent a starting point for the study of a possible combined treatment (atmospheric pressure cold plasmas and a MDR efflux pumps inhibitor applied with chemotherapy).

The novel arthritis-drug substance MCS-18 attenuates the antibody production in vivo.

UNLABELLED: Influence of the novel arthritis drug-substance MCS-18 on the antibody (Ab) production against tetanus toxoid (TT) and diphtheria toxoid (DT) antigens was tested in vivo. Possible involvement of MCS-18 in Toll-like receptor (TLR) signalling pathway was further considered. MATERIALS AND METHODS: Immunization of male CD1 mice was done with subcutaneous injection of TT emulsified in Freund's Complete (FCA) or Incomplete Adjuvant (FIA) and mixed diversly with MCS-18 and different test substances. To investigate the influence of TLR activation Pam3Cys and lipopolysaccharide (LPS) emulsified in FIA were tested in combinations with MCS-18. Antibody production was analysed in vivo by tetanus- or diphtheria-toxin neutralization test. RESULTS: Immunogenicity of TT was significantly enhanced if administered together with FCA or TLR agonists Pam3Cys or LPS emulsified in FIA. It was shown that MCS-18 attenuated strongly the production of anti-TT Ab if administered together with the Ab elicitor FCA or TLR agonists in various combinations. MCS-18 was also active via oral administration. DISCUSSION: These findings suggest that MCS-18 could be a potent, non-toxic antagonist or a down-regulator of TLR signalling pathway. Investigations on further models are needed to establish ifMCS-18 may influence particularly the production of RA-specific auto-antibodies, too.

Study of apoptosis induced by cytostatics and vegetal extracts on human endothelial cell line.

Angiogenesis, the biological process by which new capillaries are formed from pre-existing vessels, is a tightly controlled and complex process involving several factors with both stimulating and inhibiting steps. In solid tumor growth, a specific clinical turning point is the transition to the vascular phase. Once it develops an intrinsic vascular network, a tumor grows indefinitely. Tumor angiogenesis depends mainly on the release by neoplasic cells of growth factors specific for endothelial cells (ECs), able to stimulate growth of the host blood vessels. The aim of this study was to analyze the apoptotic effect of some cytostatics, Vinblastine, Rapamycin and Doxorubicin, and vegetal extracts (called VOB) isolated and purified from Vitis sp., on human EA.hy926 endothelial cell line. In a proliferation assay using Crystal Violet, we demonstrated that Vinblastine and Rapamycin cytostatics have synergistic effect on endothelial cell line EA.hy926 growth inhibition. The inhibitory effects of Vinblastine and Doxorubicin were enhanced by VOB vegetal extracts. A combined treatment of cytostatics and VOB vegetal extracts resulted in a stronger antiproliferative effect of EA.hy926 endothelial cells. Results obtained regarding the apoptosis induced on EA.hy926 endothelial cells showed that each compound alone was able to induce a significant percent of apoptotic cells in a dose-dependent manner.

COX-2 inhibitors can down-regulate in vivo antibody response against T-dependent antigens.

There are many studies demonstrating by different experimental models that non-steroidal antiinflammatory drugs (NSAIDs), also known as cyclooxygenase-2 (COX-2) inhibitors, can modulate immune response such as lymphoid cells differentiation and proliferation. There are experimental data which show that activated B cells can express mRNA COX-2, release prostaglandins (PGs) and produce immunoglobulins in PGs dependent manner. In this study, using different COX-2 inhibitors and applying personalized immunization scheme, we confirmed that it is possible to modulate in vivo antibody response against T cell dependent antigens, substantiating the importance of PGE2 and E prostanoid receptor (EP-R) in antibody generation. Our results point out the fact that we must be more careful when we apply vaccines containing T-cell dependent antigens, such as tetanus or diphteric anatoxin, to the patients under an intense antiinflammatory treatment.

The effect of the plasma needle on tumoral cell lines apoptosis.

In this paper, we present the effect of the plasma needle on tumor cell surface. The plasma is generated at the tip of a metal needle by using a radio-frequency generator of 13.56 MHz, 100's V amplitude. In our study we investigated the interaction of non-thermal plasma (plasma needle) with living monolayer tumour cells in culture medium. We applied short needle to sample distance (1 mm) at temperature of 25 degrees C, 30 degrees C and 37 degrees C, respectively. Our data sugest that the plasma needle reduces the viability and induces apoptosis of tumour cells. These activities may be very useful in dermatology, where a part of the tissue must be removed with high-precision, without damage to the adjacent cells and without inflammatory reaction.

Antioxidant properties of some hydroalcoholic plant extracts with antiinflammatory activity.

The hydroalcoholic extracts of Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra which exhibited different anti-inflammatory activities were evaluated for the possible mode of action by studying their antioxidant potential. In the present study we investigated if standardized hydroalcoholic extracts of plants such as Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra produced by Hofigal Stock Company could modulate the respiratory burst of human activated neutrophils, as a consequence of their antioxidant capacity. Their antioxidant properties were measured using a colorimetric assay (Total Antioxidant Status kit). We demonstrated that Hypericum perforatum and Calendula officinalis hydroalcoholic extracts possessed a significant antioxidant activity while Plantago lanceolata and Glycyrrhiza glabra hydroalcoholic extracts had a minor antioxidant status. Using reactive oxygen species-generating systems (OZ-activated human PMN neutrophils), Calendula officinalis and Hypericum perforatum extracts showed strong reactive oxygen species scavenging property, Hypericum perforatum extract exhibing the highest scavenging activity. These results confirm the potential of Calendula officinalis and Hypericum perforatum investigated hydroalcoholic extracts as medicinal remedies to be used in different inflammatory/allergic diseases. These extracts could be a useful tool for obtaining new antioxidant/anti-inflammatory agents.

Hydroalcoholic plant extracts with anti-inflammatory activity.

The aim of the present study was to investigate if standardized hydroalcoholic plant extracts such as Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra can suppress in cell-free systems the activities of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2), key enzymes in the formation of proinflammatory eicosanoids from arachidonic acid (AA). Studies were undertaken to compare the above mentioned plant extracts to a known NSAID (nimesulide) in their ability to inhibit both cyclooxygenase (COX-2) and lipoxygenase (5-LO) activities in cell-free systems. We report on 2 vegetal extracts (Hypericum perforatum and Glycyrrhiza glabra) that inhibit 5-LO activity and 2 vegetal extracts (Plantago lanceolata and Glycyrrhiza glabra) that inhibit COX-2 activity. In this study, we demonstrate for the first time that Glycyrrhiza glabra extract efficiently suppresses both eicosanoids and leukotrienes formation in cell-free systems, implying that this extract directly acts as a dual inhibitor of 5-LO and COX-2 activities. With regard to the properties of dual COX-2/5-LO inhibitors, Glycyrrhiza glabra extract might be a potential drug possessing anti-inflammatory activity devoid of the most troublesome (gastric) side effects seen for drugs used as COX-2 and 5-LO inhibitors. Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra extracts can be added to an already impressive list of these species that have anti-inflammatory activity.