Nanoparticle delivery of grape seed-derived proanthocyanidins to airway epithelial cells dampens oxidative stress and inflammation.

BACKGROUND: Chronic respiratory diseases, whose one of the hallmarks is oxidative stress, are still incurable and need novel therapeutic tools and pharmaceutical agents. The phenolic compounds contained in grape are endowed with well-recognized anti-oxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Considering that natural anti-oxidants, such as proanthocyanidins, have poor water solubility and oral bioavailability, we have developed a drug delivery system based on solid lipid nanoparticles (SLN), apt to encapsulate grape seed extract (GSE), containing proanthocyanidins. METHODS: Plain, 6-coumarin (6-Coum), DiR- and GSE-loaded SLN were produced with the melt-emulsion method. Physicochemical characterization of all prepared SLN was determined by photon correlation spectroscopy and laser Doppler anemometry. MTT assay (spectrophotometry) and propidium iodide (PI) assay (cytofluorimetry) were used to assess cell viability. Flow cytometry coupled with cell imaging was performed for assessing apoptosis and necrosis by Annexin V/7-AAD staining (plain SLE), cell internalization (6-Coum-SLN) and reactive oxygen species (ROS) production (SLN-GSE). NF-κB nuclear translocation was studied by immunofluorescence. In vivo bio-imaging was used to assess lung deposition and persistence of aerosolized DiR-loaded SLN. RESULTS: Plain SLN were not cytotoxic when incubated with H441 airway epithelial cells, as judged by both PI and MTT assays as well as by apoptosis/necrosis evaluation. 6-Coum-loaded SLN were taken up by H441 cells in a dose-dependent fashion and persisted into cells at detectable levels up to 16 days. SLN were detected in mice lungs up to 6 days. SLN-GSE possessed 243 nm as mean diameter, were negatively charged, and stable in size at 37 °C in Simulated Lung Fluid up to 48 h and at 4 °C in double distilled water up to 2 months. The content of SLN in proanthocyanidins remained unvaried up to 2 months. GSE-loaded SLN determined a significant reduction in ROS production when added 24-72 h before the stimulation with hydrogen peroxide. Interestingly, while at 24 h free GSE determined a higher decrease of ROS production than SLN-GSE, the contrary was seen at 48 and 72 h. Similar results were observed for NF-κB nuclear translocation. CONCLUSIONS: SLN are a biocompatible drug delivery system for natural anti-oxidants obtained from grape seed in a model of oxidative stress in airway epithelial cells. They feature stability and long-term persistence inside cells where they release proanthocyanidins. These results could pave the way to novel anti-oxidant and anti-inflammatory therapies for chronic respiratory diseases.

Immunopathogenesis of neurodegenerative diseases: current therapeutic models of neuroprotection with special reference to natural products.

Parkinson disease (PD) and Alzheimer disease (AD) are neurodegenerative processes whose frequency is dramatically increasing in the western world. Both diseases share a common pathogenic denominator characterized by an exaggerated activation of the systemic and cerebral immune system, respectively. For instance, lipopolysaccharides in PD and amyloid beta in AD trigger microglia and astrocytes to release reactive oxygen species (ROS) and proinflammatory cytokines. Infiltrating peripheral T cells once activated in the central nervous system also contribute to the neurodegenerative process. Besides innovative biotherapy, nutraceuticals or functional foods are currently investigated for their neuroprotective activities. Especially, vitamin D and polyphenols, seem to be promising therapeutic tools for inhibiting ROS formation and arresting cytokine-mediated neuroinflammation in PD and AD.

Fermented grape marc (FGM): immunomodulating properties and its potential exploitation in the treatment of neurodegenerative diseases.

The onset of neurodegenerative diseases has become more frequent than in the past also in relation to inappropriate dietary habits adopted in the western world. Nutraceuticals are currently investigated in order to prevent or retard the outcome of the so-called diet-related diseases, even including neurodegenerative pathologies. Here, we have in vitro studied the ability of fermented grape marc (FGM) from Negroamaro (N) and Koshu (K) Vitis vinifera to modulate the function of human peripheral blood mononuclear cells (PBMCs). Actually, both FGMs were able to increase the release and the intracellular content of inflammatory and anti-inflammatory cytokines, the induction of FoxP3 (a biomarker of T regulatory cells) and reduce the production of Granzyme B from PBMCs. Since these FGM-induced effects tend to polarize the immune response toward an anti-inflammatory pathway, the potential use of FGMs may represent a valid therapeutic measure to mitigating neuroinflammation in pathologies such as Parkinson disease and Alzheimer disease.

The impact of bacterial lipolysaccharides on the endothelial system: pathological consequences and therapeutic countermeasures.

Endothelial cells (ECs) express on their membrane Toll like receptor (TLR)-4 and, therefore, are able to interact with lipopolysaccharides (LPS) or endotoxins, major constituents of the gram-negative bacteria outer membrane. The impact of LPS on ECs can be either direct or mediated via release of cytokines and/or chemokines originated from monocytes/macrophages. In this review, the effect of the interaction between LPS and ECs on the outcome of various human diseases such as preeclampsia, hereditary haemorrhagic teleangiectasia, atherosclerosis and sepsis will be illustrated. Finally, the major therapeutic attempts aimed at neutralizing LPS and, therefore, their influence on ECs will be discussed.

Potential application of dietary polyphenols from red wine to attaining healthy ageing.

Polyphenols are ubiquitous compounds present in the vegetal kingdom and endowed with an array of beneficial activities to human health. In this review, the effects of dietary polyphenols on the prevention and/or mitigation of cancer, neurodegenerative diseases, obesity, metabolic syndrome and atherosclerosis will be illustrated. Moreover, emphasis will be placed on our own data concerning the in vitro effects performed by polyphenols from an Italian red wine "Negroamaro" on human healthy peripheral blood mononuclear cells. Particularly, production of nitric oxide and maintenance of the inflammatory/anti-inflammatory cytokine network will be discussed also in relation to potential application to human age-related diseases. In conclusion, polyphenols in virtue of the plethora of protective effects manifested in various experimental models and clinical trials seem to be appropriate as dietary supplements for preventing the functional decline of organs with age.

Low grade inflammation as a common pathogenetic denominator in age-related diseases: novel drug targets for anti-ageing strategies and successful ageing achievement.

Nowadays, people are living much longer than they used to do, however they are not free from ageing. Ageing, an inexorable intrinsic process that affects all cells, tissues, organs and individuals, is a post-maturational process that, due to a diminished homeostasis and increased organism frailty, causes a reduction of the response to environmental stimuli and, in general, is associated to an increased predisposition to illness and death. However, the high incidence of death due to infectious, cardiovascular and cancer diseases underlies a common feature in these pathologies that is represented by dysregulation of both instructive and innate immunity. Several studies show that a low-grade systemic inflammation characterizes ageing and that inflammatory markers are significant predictors of mortality in old humans. This pro-inflammatory status of the elderly underlies biological mechanisms responsible for physical function decline and age-related diseases such as Alzheimer's disease and atherosclerosis are initiated or worsened by systemic inflammation. Understanding of the ageing process should have a prominent role in new strategies for extending the health old population. Accordingly, as extensively discussed in the review and in the accompanying related papers, investigating ageing pathophysiology, particularly disentangling age-related low grade inflammation, is likely to provide important clues about how to develop drugs that can slow or delay ageing.

Donkey's and goat's milk consumption and benefits to human health with special reference to the inflammatory status.

The environmental impact as well as malnutrition are responsible for an increased incidence of inflammatory diseases with a consequential loss of immune homeostasis. Therefore, administration of nutraceuticals is aimed at reconstituting the immune balance in terms of the so-called immune nutrition. Among many known nutraceuticals, more recently, donkey's and goat's milks have been used as good alternatives to human and bovine milk in various clinical conditions such as allergy, atopy and inflammatory diseases. In fact, both milks possess immunomodulating capacities and release nitric oxide, a potent vasodilator endowed with anti-atherogenic properties. In this review, emphasis will be placed on the consumption of fermented milk and, in particular, on its ability to modulate the aged immune system, even including the intestinal mucosal immune response in elderly. Therefore, for their specific properties donkey's and goat's milk administration to aged people should be encouraged.

Ability of goat milk to modulate healthy human peripheral blood lymphomonocyte and polymorphonuclear cell function: in vitro effects and clinical implications.

The in vitro effects of goat's milk from different sources (Jonica, Saanen, and Priska breeds plus a commercial preparation) on healthy human peripheral blood mononuclear cells (PBMCs) were evaluated in terms of nitric oxide (NO) and cytokine release. According to the incubation time (24 h or 48 h) used all milks could induce release of NO from monocytes. In this context, however, in the presence of a commercial milk preparation inhibition of lypopolysaccharide (LPS)-induce NO generation was evident. Also polymorphonuclear cells stimulated with the various milks released detectable amounts of NO. In the case of Priska milk inhibition of LPS-mediated NO generation was observed. Despite a broad array of cytokines tested [Interleukin (IL)-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, Tumor Necrosis Factor (TNF)-alpha, Transforming Growth Factor-beta and Granulocyte Colony Stimulating Factor] only IL-10, TNF-alpha, and IL-6 were released by PBMCs upon stimulation with various milks. Taken together, these data indicate that goat's milk for its capacity to produce NO may exert a cardioprotective and anti-atherogenic effect in consumers. Moreover, induction of proinflammatory (TNF-alpha and IL-6) and anti-inflammatory (IL-10) cytokines suggests the ability of this milk to maintain immune homeostasis in the immunocompromised host (e.g., aged people).

A broad variety of antigens contribute to the pathogenesis of atherosclerosis: how to neutralize noxious reactions in the host.

Atherosclerosis is an inflammatory disease characterized by lipid accumulation as well as vascular injury due to a massive infiltration of immune cells in the endothelial wall. Microbial and self- antigens are responsible for a persistent activation of immune and non-immune cells, thus leading to a condition of arterial chronic inflammation with plaque formation and rupture in complicated cases. In particular, the effects of bacteria, viruses and their toxic products as well as of glycated lipoproteins will be illustrated with special reference to the main inflammatory pathways triggered by a persistent antigenic load in the host. Taking into consideration this broad variety of antigens implicated in the pathogenesis of atherosclerosis, therapeutic approaches such as antibiotics, dietary intervention and immune therapies will be discussed.

A scientific approach to anti-ageing therapies: state of the art.

A lasting dream of human beings is to reverse or at least postpone ageing. During the last years, an increasing number of scientific meetings, articles, and books have been devoted to anti-ageing therapies. This subject, full of misleading, simplistic, or wrong ideas, is very popular among the general public, whose imagery has been fascinated by all possible tools to delay ageing, getting immortality. Here, we discuss anti-ageing strategies aimed not to rejuvenate but to slow ageing and delay the onset of age-related diseases. These approaches should be able to substantially slow down the ageing process, extending our productive, youthful lives.

Polyphenols from red wine modulate immune responsiveness: biological and clinical significance.

Many studies have been conducted on the effects of red wine polyphenols on certain diseases, primarily, coronary heart disease (CHD) and, in this respect, evidence has been demonstrated that intake of red wine is associated with a reduction of CHD symptomatology. In this framework, the purpose of this review is to illustrate the effects of polyphenols on immune cells from human healthy peripheral blood. Data will show that polyphenols are able to stimulate both innate and adaptive immune responses. In particular, the release of cytokines such as interleukin (IL)-12, interferon (IFN)-gamma, and IL-10 as well as immunoglobulins may be important for host protection in different immune related disorders. Another important aspect pointed out in this review is the release of nitric oxide (NO) from peripheral blood mononuclear cells (PBMC), stimulated by red wine polyphenols despite the fact that the majority of studies have reported NO production only by endothelial cells. Release of NO from PBMC may play an important role in cardiovascular disease, because it is known that this molecule acts as an inhibitor of platelet aggregation. On the other hand, NO exerts a protective role against infectious organisms. Finally, some molecular cytoplasmatic pathways elicited by polyphenols able to regulate certain immune responses will also be discussed. In particular, it seems that p38, a molecule belonging to the MAPK family, is involved in the release of IFN-gamma and, therefore, in NO production. All these data confirm the beneficial effects of polyphenols in some chronic diseases.

Elicitation of immune responsiveness against antigenic challenge in age-related diseases: effects of red wine polyphenols.

Polyphenols contained in red wine possess a broad array of properties which seem to be beneficial to human and animal health. We have investigated the ability of red wine polyphenols to promote the in vitro release of both proinflammatory and antiinflammatory cytokines from human healthy mononuclear cells, as well as of immunoglobulins from B cells. Following red wine (Negroamaro) pretreatment of lymphomonocytes, results will show a production of regulatory [Interleukin(IL)-12], proinflammatory (IL-1 beta and IL-6), and anti-inflammatory (IL-10) cytokines, as well as of IgA and IgG. The fine balance between inflammation and antiinflammation, as well as the role of humoral immune response either systemic or mucosal will be discussed as a consequence of red wine intake. Finally, since ageing is characterized by a decline of many immune functions, our results suggest that moderate use of red wine may be beneficial in age-related disorders where the host immune response is very often not effective against a variety of antigens.

Molecular effects elicited in vitro by red wine on human healthy peripheral blood mononuclear cells: potential therapeutical application of polyphenols to diet-related chronic diseases.

Red wine represents a source of polyphenols which exhibit a number of biological effects on various systems. In this respect, there is evidence that red wine polyphenols constitute one of the ingredients of the Mediterranean diet which is associated to a reduced risk of coronary heart disease according to current literature. Here, we have evaluated in vitro the molecular mechanisms elicited by polyphenols from red wine (Negroamaro) on human healthy mononuclear cells. In particular, we have investigated the involvement of polyphenols in the activation of p38 and ERK1/2 molecules belonging to the MAPK kinase family and on the expression of I kappaB alpha and p65/NF kappaB. Results will demonstrate that in cells both the expression of p38 and ERK1/2 augments in the presence of red wine polyphenols, but their expression drops in the presence of polyphenols plus lipopolysaccharides (LPS). This indicates that in Gram-negative infections polyphenols may attenuate triggering of inflammatory mediators as a response to LPS stimulation. Finally, the regulatory role of polyphenols on I kappaB alpha and p65/NF kappaB expression is discussed, pointing out that red wine might favor anti-atherogenic mechanisms in the course of cardiovascular disease.

Immunological properties of donkey's milk: its potential use in the prevention of atherosclerosis.

Donkey's milk is the best substitute of human milk for its content in lactose, proteins, minerals, and omega-3 fatty acids. Here, we have evaluated the effects of colostrum and milk from donkeys (Martina Franca breed) on the function of human peripheral blood mononuclear cells (PBMCs) at different intervals from lactation. Colostrum induced more IgA responses, while milk induced predominantly more IgG responses. Both milk and colostrum induced expression of CD25 and CD69 on PBMCs. The ability to induce release of interleukins (IL) (IL-12, IL-1 beta and IL-10) and tumor necrosis factor-alpha was confined only to milk, while colostrum was devoid of this capacity. Finally, both colostrum and milk induced nitric oxide (NO) release from PBMCs but milk exhibited a greater capacity than colostrum in NO generation. Taken together, these immunological activities exerted by both colostrum and milk from donkeys may be useful in the treatment of human immune-related diseases. In particular, NO induction by donkey's milk may be very useful in the prevention of atherosclerosis, being a strong vasodilator and an effective antimicrobial agent since pathogens and/or their products may play a proatherogenic role.

Red wine consumption and prevention of atherosclerosis: an in vitro model using human peripheral blood mononuclear cells.

Evidence has been provided that red wine possesses antiatherogenic activities in virtue of its content in polyphenols (flavonoids and non-flavonoids substances). Here, some red wines (Negroamaro, Primitivo and Lambrusco) were tested for their ability to trigger nitric oxide (NO) production from human healthy peripheral blood mononuclear cells (PBMC). Negroamaro was the strongest inducer of NO from PBMC and deprivation of polyphenols did not influence its NO generation capacity. This fact supports the involvement of polyphenols in the NO production even in the absence of alcohol, which also per se does not exert any significant activity. These results are also corroborated by the evidence that PBMC inducible-nitric oxide synthase expression occurred by the effect of samples containing polyphenols but this expression was very weak when polyphenols were removed from the whole Negroamaro. In synthesis, flavonoids and resveratrol, major constituents of red wine, once absorbed at intestinal level, enter circulation and trigger monocytes for NO production. To the best of our knowledge, this is the first demonstration of a direct effect of red wine on monocytes for NO release to occur. On the other hand, also the macrophage contingent from gut-associated lymphoid tissue can contribute to NO generation, besides the aliquot produced by endothelial cells, as previously demonstrated by various authors. Taken together, these results support the concept that moderate intake of red wine can prevent atherosclerosis via production of NO, a potent vasodilator of terminal vessels.

Immune abnormalities and endotoxemia in patients with ulcerative colitis and in their first degree relatives: attempts at neutralizing endotoxin-mediated effects.

Proinflammatory cytokines released from monocytes/macrophages, in particular tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, and IL-8 seem to play an important role in Inflammatory Bowel Disease (ulcerative colitis and Crohn's disease). Endotoxins or lipopolysaccharides, derived from the outer membrane of Gram-negative bacteria interact with CD14 on surface membrane of macrophages, thus triggering a signal cascade, which leads to the production and release of proinflammatory cytokines, particularly TNF-alpha. Therefore, in IBD, lipopolysaccharides could play a pathogenic role. In this respect, plasma endotoxins have been demonstrated in a not negligible percentage of patients with ulcerative colitis and in their unaffected relatives. The presence of circulating endotoxins could be due, at least in part, to the impaired natural immunity in either patients with ulcerative colitis or in their first degree unaffected relatives. Lactoferrin is an iron-binding glycoprotein, which binds to the lipid A region of lipopolysaccharide with a high affinity and this interaction prevents the binding of lipopolysaccharide to CD14, thus inhibiting the release of proinflammatory cytokines. Therefore, based on the possible pathogenic role exerted by endotoxins in ulcerative colitis, lactoferrin may deserve attention as a possible therapeutical agent in experimental models of Inflammatory Bowel Disease.

Modifications of the immune responsiveness in patients with autoimmune thyroiditis: evidence for a systemic immune alteration.

Hashimoto's thyroiditis, the most common form of autoimmune thyroid disease, is characterised by lymphocytic infiltration of the thyroid gland, gradual destruction of the organ and production of thyroid specific auto antibodies (antithyroid peroxidase and antithyroglobulin antibodies). There are evidences that cast doubt on the pathogenetic role of these antibodies in thyroid autoimmunity. It is very likely that cellular destruction is mediated by other cellular mechanisms, such as auto reactive T-lymphocytes, natural killer and cytokines. However, other studies performed in animal models have led to the conclusion that organ specific autoimmune thyroiditis should be regarded as a polygenic disease with a penetrance that is strongly influenced by environmental factors. According to our recent results, patients affected by autoimmune thyroiditis exhibited a decreased percentage of NK and CD25 + bearing cells significantly in comparison to normal controls. Altogether these data indicated that in the patients with autoimmune thyroid disease a certain degree of peripheral immune deficiency was present.

Modifications of the immune responsiveness in patients with hepatitis C virus infection following treatment with IFN-alpha/ribavirin.

The balance between T helper (h)1 and Th2 responsiveness seems to represent a key event in the evolution of hepatitis C virus (HCV) infection. In particular, Th1 cytokines [interleukin (IL-2) and interferon (IFN-gamma)] have been demonstrated to mediate the antiviral immune response. Serum levels of Th1 cytokines (IL-2 and IFN-gamma) as well as of Th2 products (IL-4 and IL-10) were determined in a group of HCV-positive patients before and after treatment with IFN-alpha and Ribavirin (RIB). Results indicate that responder patients exhibited increased levels of IFN-gamma and IL-10, while this enhancement was not observed in non-responder patients. In this respect, the major effect exerted by the combined therapy with IFN-alpha/RIB could be represented by the attainment of a re-equilibrium between inflammatory (Th1) and antiinflammatory (Th2) mechanisms. In this framework, according to current literature, novel therapeutical approaches to treat HCV infection are represented by administration of recombinant IL-2 and IL-10.

Biological and clinical significance of endotoxemia in the course of hepatitis C virus infection.

Endotoxins or lipopolysaccharides (LPS), major components of the cell wall of Gram-negative bacteria, once released from the bacterial outer membrane bind to specific receptors and, in particular, to a membrane-bound receptor, the CD14 (mCD14) and the toll-like receptor 4 present on monocytes/ macrophages. In turn, LPS-activated monocytes/ macrophages release in the host tissue an array of so-called proinflammatory cytokines and, among them, Tumor Necrosis Factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8 and IL-12 are the major mediators. Before therapy (To) and at the end of 6-month interferon (IFN)-alpha/Ribavirin (RIB) treatment (T6), circulating endotoxin levels were measured in responder and non responder HCV+ patients. At T0, 57% of the non responders were endotoxin-positive and had, on average, 54 pg/ml of plasma LPS while in 50% of the responder patients endotoxin were found with an average of 29 pg/ml. At T6, in responders LPS were no longer detectable, while in 42% of the non responders LPS were found (average levels 45 pg/ml). In terms of serum cytokine concentration, at T6 IFN-gamma levels when compared to those detected at T0 were increased in both endotoxin-positive and endotoxin-negative patients. However, at T6 IL-10 concentration was significantly increased only in the group of endotoxin-negative subjects (responder patients), in comparison to T0 values. The origin of endotoxemia in HCV+ patients seems to be multifactorial, likely depending on impaired phagocytic functions and reduced T-cell mediated antibacterial activity. In these patients, however, one cannot exclude the passage of LPS from the gut flora to the blood stream, owing a condition of altered intestinal permeability. At the same time, a less efficient detoxification of enteric bacterial antigens at the hepatic level should be taken into consideration. Finally, novel therapeutic attempts aimed to neutralize LPS in the host are discussed.

The role of the liver in the response to LPS: experimental and clinical findings.

The liver plays an important physiological role in lipopolysaccharide (LPS) detoxification and, in particular, hepatocytes are involved in the clearance of endotoxin of intestinal derivation. In experimental shock models, tumor necrosis factor (TNF)-alpha induces hepatocyte apoptosis and lethal effects are due to secreted TNF-alpha and not to cell-associated TNF-alpha. An exaggerated production of TNF-alpha has been reported in murine viral infections, in which mice become sensitized to low amounts of LPS and both interferon (IFN)-gamma and IFN-alpha/beta are involved in the macrophage-induced release of TNF-alpha. The prominent role of LPS and TNF-alpha in liver injury is also supported by studies of ethanol-induced hepatic damage. In humans, evidence of LPS-induced hepatic injury has been reported in cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis and a decreased phagocytic activity of the reticulo-endothelial system has been found in these diseases. The origin of endotoxemia in hepatitis C virus (HCV) infected patients seems to be multifactorial and LPS may be of exogenous or endogenous derivation. In endotoxemic HCV-positive patients responsive to a combined treatment with IFN-alpha/ribavirin (RIB), endotoxemia was no longer detected at the end of the therapeutic regimen. By contrast, 48% of the non-responders to this treatment were still endotoxemic and their monocytes displayed higher intracellular TNF-alpha and interleukin (IL)-1beta levels than responders. Moreover, in responders, an equilibrium between IFN-gamma and IL-10 serum levels was attained. In the non-responders, serum levels of IL-10 did not increase following treatment. This may imply that an imbalance between T helper (Th)1 and Th2 derived cytokines could be envisaged in the non-responders.