Primary Prevention of Alzheimer’s Disease (AD)

Alzheimer dementia (AD) is a complex, aging-associated disease whose effects on the brain (an organ made up by nonreplaceable cells) are devastating. Disease is not curable, but progress in pathobiology shows that intervention on aging can make primary prevention of AD feasible. According to the amyloid-cascade hypothesis, mechanisms of AD include: an age-related alteration of free radical metabolism in membranes, leading to a higher yield in the toxic Aβ1-42 peptide and an overwhelming impact on the weaker repair mechanisms of the aging cells. The proposed intervention on aging with anti-AD effects includes a daily assumption of antioxidants (red wine polyphenols enriched with resveratrol), a reinforcement of membrane antioxidant defenses by the assumption of polyunsaturated fatty acids at the first meal after fasting, and an enhancement of cell repair function (at the proteasome and autophagy level by an intermittent feeding regimen and physical exercise plus the assumption of antilipolytic agents during time of fasting). The beneficial effects of diet and physical activity on the endogenous production of protective nerve growth factors are magnified by an enriched environment. Treatment has already been started on healthy individuals at a higher risk of AD in the city of Volterra

Identification of Pseudomonas aeruginosa released proteins: effects of oxygen limitation and azithromycin treatment in clinical and laboratory strains

Caratteristica della fibrosi cistica (FC) \ue8 la colonizzazione del polmone da parte di batteri, in particolare Pseudomonas aeruginosa (Pa). Essa prevede l\u2019instaurarsi di un\u2019interazione batterio-ospite, la quale \ue8 mediata non solo dal contatto diretto con il microorganismo, solitamente ostacolato dalla presenza di dense secrezioni e dall\u2019insediamento di ceppi mucoidi, ma anche dal rilascio di fattori specifici. Pa cresce nelle vie respiratorie di pazienti FC in condizioni di microaerobiosi. Quando le cellule epiteliali delle vie respiratorie FC sono state trattate con surnatante batterico ottenuto in seguito alla cultura del ceppo clinico AA2, cresciuto in aerobiosi e microaerobiosi, si \ue8 osservato in entrambi le condizioni un significativo aumento di IL-8 mRNA, a differenza del surnatante del ceppo di laboratorio PAO1 che non ha indotto alcuna significativa risposta pro-infiammatoria. Allo scopo di individuare fattori proteici rilasciati da Pa aventi attivit\ue0 pro-infiammatoria \ue8 stata applicato un approccio proteomico innovativo (MudPIT: Multidimensional Protein Identification Technology) che ha permesso di identificare 451 e 235 proteine nel surnatante di AA2 e PAO1 rispettivamente. L\u2019analisi proteomica ha rivelato che varie proteine sono espresse e rilasciate in modo differente a seconda del ceppo, clinico o di laboratorio, e delle condizioni di coltura, aerobiche o microaerobiche. Tra queste, di particolare interesse, vi sono undici diverse proteasi che sono state trovate nel surnatante del ceppo AA2, mentre solo quattro sono stati rilevate nel surnatante del ceppo PAO1. Ecotin, un inibitore delle proteasi, \ue8 presente in quantit\ue0 notevole nel surnatante di PAO1 rispetto a quello di AA2 ottenuti entrambi in microaerobiosi. Questi risultati sono stati confermati dai saggi di Western blot e zimogramma. Il livello di espressione delle proteasi e dell\u2019inibitore ecotin nei due ceppi correla con l\u2019attivit\ue0 pro-infiammatoria osservata in vitro. Solo nel 31% dei ceppi di Pseudomonas aeruginosa isolati da pazienti FC cronicamente infetti \ue8 stata osservata una discreta attivit\ue0 metalloproteasica mentre \ue8 state identificata in tutti gli isolati che derivano da pazienti FC infettati sporadicamente. Questi risultati suggeriscono che la tecnologia MudPIT pu\uf2 essere utile per dipanare la complessit\ue0 del microambiente polmonare caratterizzato da infezione e infiammazione cronici e per facilitare l'identificazione delle molecole chiave coinvolte nell\u2019interazione tra batterio Pa e vie respiratorie. Negli ultimi anni c\u2019\ue8 stato un crescente interesse per l'utilizzo dell\u2019antibiotico azitromicina (AZM) per il trattamento delle patologie polmonari in pazienti affetti da fibrosi cistica. L\u2019azitromicina agisce quale inibitore della sintesi proteica nei batteri. Nonostante Pseudomonas aeruginosa risulti essere resistente all\u2019azione del farmaco, la somministrazione del macrolide comporta in soggetti affetti da infezione cronica un miglioramento dell\u2019intero quadro patologico. E\u2019 stato suggerito che la modulazione dei fattori di virulenza di Pa sia correlata agli effetti benefici riscontrati nei pazienti FC. Abbiamo testato gli effetti dell\u2019 azitromicina in relazione all\u2019 isolato clinico AA2, per stabilire come questo farmaco potrebbe interferire con la produzione dei fattori di virulenza batterica. Abbiamo dimostrato che l'aumento di IL-8 mRNA in cellule epiteliali CF indotto dal surnatante prodotto dalla coltura di AA2 \ue8 stato significativamente ridotto quando il ceppo clinico \ue8 stato cresciuto in presenza di AZM, suggerendo che questo macrolide riduce la patogenicit\ue0 di Pa. Nel tentativo di ottenere informazioni sull'identit\ue0 delle molecole rilasciato dal ceppo clinico PA prima e dopo il trattamento con AZM abbiamo applicato la tecnologia MudPIT. Abbiamo trovato 12 proteine differenzialmente espresse e rilasciate nel surnatante di AA2 cresciuto in presenza e assenza dell\u2019antibiotico. Una delle proteine di interesse \ue8 la metalloproteasi alcalina (APR) che viene down-regolata in presenza di AZM. E\u2019 stato anche osservato che AZM diminuisce l'attivit\ue0 metalloproteasica e l\u2019espressione di APR in surnatanti di isolati clinici di individui infettati sporadicamente, mentre nessun effetto \ue8 stato osservato nei surnatanti isolati di pazienti FC cronicamente infetti. Questi risultati sono stati validati per mezzo di zimogramma e Western blot. L'analisi MudPIT delle proteine rilasciate dal ceppo clinico AA2 cresciuto in assenza e in presenza di AZM suggerisce che tale macrolide possa diminuire l'espressione e il rilascio di fattori associati alla virulenza del batterio. Gli effetti dell\u2019 AZM, inoltre, sull'espressione e il rilascio di fattori pro-infiammatori da ceppi clinici possono contribuire a spiegare i benefici clinici associati con la terapia del macrolide.Colonization by Pseudomonas aeruginosa (Pa) is a hallmark of lung disease in cystic fibrosis (CF), where microaerobic conditions develop as a consequence of disease progression. Conditioned medium (CM) obtained from Pa clinical strain AA2, unlike the CM from laboratory strain PAO1, induces in airway epithelial cells IL-8 mRNA in both aerobic and microaerobic conditions. The effect was impaired by protease digestion. Shotgun proteomic analysis (Multidimensional protein identification technology: MudPIT) of conditioned medium of PAO1 and AA2 identified 451 and 235 individual proteins. Various proteins were found differentially regulated between strains and culture conditions. Among these eleven different proteases were found released by the AA2 strain, while fewer peptides of only four of them were detected in the PAO1 strain. Ecotin, a protease inhibitor, was found to be highly represented in PAO1 in comparison with AA2 grown in microaerobiosis. These results were confirmed by functional assay (zymography) and western blotting. The pattern of expression of several proteases and their inhibitor ecotin correlates with pro-inflammatory activity in vitro better than other candidate virulence factors. Only 31% of the Pa strains isolates from chronically infected CF patients expressed detectable metalloprotease activity while all the isolates derived from sporadically infected individuals scored positive (individual strains analyzed: 42, p<0.002). These results suggest that high-throughput approaches are critical to unravel the complexity of the pro-inflammatory microenvironment associated to the presence of Pa and to facilitate the identification of key molecules involved in Pa biology/pathology. There is considerable interest in the use of azithromycin (AZM) for the treatment of lung disease in patients with cystic fibrosis. Although its mechanism of action as an inhibitor of bacterial protein synthesis has been well established, it is less clear how AZM ameliorates the lung disease associated with P. aeruginosa, which is considered to be resistant to the drug. Modulation of Pa virulence factors was suggested as mechanism for AZM beneficial effects in CF patients. We tested the effects of azithromycin on clinical isolate AA2 to establish how this drug might interfere with the production of bacterial virulence factors that are relevant to the pathogenesis of airway disease in CF patients. We demonstrated that the increase of IL-8 mRNA in CF epithelial cells induced by CM from AA2 was significantly reduced when the clinical strain was grown in the presence of AZM, suggesting that this macrolide reduces Pa pathogenicity. In the attempt to gain information on the identity of the molecules released by Pa clinical strain before and after treatment with AZM we applied MudPIT. We found 5 upregulated and 7 downregulated proteins in CM from AA2 incubated with AZM. Peptides from the alkaline metalloproteinase precursor (APR) were less represented in CM derived from AA2 strain grown in presence of AZM than in those from the same strain cultured in absence of this macrolide. AZM was observed also to decrease the metalloprotease activity and APR expression in CM of Pa isolates derived from sporadically infected individuals while any effect was detected in CM of Pa isolates from chronically infected CF patients. These results was validated by means of zymography assay and western blot technique. The MudPIT analysis of released proteins from Pa clinical isolate grown alone and in presence of AZM gives suggestion on the macrolide ability to decrease the expression of substances that contributes to Pa virulence, such as alkaline metalloproteinase. The effects of AZM on the expression and release of selected polypeptides by Pa strains may help to explain the clinical benefits associated with macrolide therapy

Resveratrol Requires Red Wine Polyphenols for Optimum Antioxidant Activity

Objective: There is substantial evidence that a diet rich in fruit and vegetables may reduce the risk of aging and stress oxidative associated diseases. It has been suggested that benefits associated with fruit and red wine consumption could be due to pooled antioxidant microcomponents in diet. The aim of this study was to investigate the antioxidant activities of pure resveratrol (a well known phytoalexin, RSV) and red wine polyphenols (RWP), using UV-B radiated isolated rat hepatocytes as a model of oxidative stress. Methods: Rat hepatocytes were isolated by the collagenase method. The cells were loaded with resveratrol and/or polyphenols at different concentrations. The production of thiobarbituric acid reactive substances (TBARS) released by UV-B radiated cells and the levels of lipid-soluble antioxidants (Dolichol, Vitamin E, Coenzyme Q9 and Q10) were measured. Results: Resveratrol had pro-oxidant or antioxidant effects depending on (lower or higher) dosage. RWP protection from photolipoperoxidation was dose-dependent and increased with dosage. Combination of the two compounds exhibited synergistic antioxidant effect, and made resveratrol effective both at lower and higher dosages. Conclusions: These results suggest that resveratrol requires red wine polyphenols for optimum antioxidant activity

Peroxisomes proliferation and pharmacological stimulation of autophagy in rat liver: evidence to support that autophagy may remove the “older” peroxisomes

Like mitochondria, peroxisomes produce reactive oxygen species (ROS), compounds which have been implicated to play an important role in many degenerative diseases and aging itself, and an exaggerated ROS production might occur in altered or older organelles. Growing evidence shows that autophagy, a required function in cell housekeeping during fasting, can remove damaged macromolecules, organelles, and membranes selectively. Proliferation of peroxisomes can be enhanced in liver cells by perfluorooctanoic acid (PFOA), which causes a marked increase of the Acyl-CoA oxidase (ACOX) activity and no significant change in urate oxidase (UOX) activity. The administration of antilipolytic drugs to fasted animals was shown to intensify autophagy. Here we tested the hypothesis that autophagy may distinguish and remove older from younger peroxisomes in rat liver. Male Sprague-Dawley rats were given PFOA (150 mg/kg body weight) or vehicle. Animals were sacrificed at different times following PFOA administration, and 3 h after the induction of autophagy with the antilipolytic agent 3,5-dimethyl pyrazole (DMP, 12 mg/kg body weight). The levels of ACOX and UOX activity were measured in the liver tissue. Results showed that autophagy caused a parallel, significant decrease in both enzymes activity in control rats, and that in PFOA-treated rats the effects were different and changed with PFOA time administration. Changes are compatible with the hypothesis that newly formed ACOX-rich peroxisomes are resistant to pexophagy and that sensitivity to pexophagy increases with increasing peroxisomal “age.” In conclusion, there is indirect evidence supporting the hypothesis that autophagy may recognize and degrade older peroxisomes

Protein Tyrosine Phosphatase Gamma (PTPγ) is a Novel Leukocyte Marker Highly Expressed by CD34+ Precursors

Protein Tyrosine Phosphatase gamma (PTPγ) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPγ is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPγ mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPγ specific antibody that recognizes the protein by flow cytometry. PTPγ expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPγ was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34+ haematopoietic precursors express high PTPγ level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPγ as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation

Inhibition of Pseudomonas aeruginosa secreted virulence factors reduces lung inflammation in CF mice

Cystic fibrosis (CF) lung infection is a complex condition where opportunistic pathogens and defective immune system cooperate in developing a constant cycle of infection and inflammation. The major pathogen, Pseudomonas aeruginosa, secretes a multitude of virulence factors involved in host immune response and lung tissue damage. In this study, we examined the possible anti-inflammatory effects of molecules inhibiting P. aeruginosa virulence factors

Indolyl aryl sulphones as HIV-1 non-nucleoside reverse transcriptase inhibitors: synthesis, biological evaluation and binding mode studies of new derivatives at indole-2-carboxamide.

New non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are active against the commonly occurring mutations of HIV are urgently needed for the treatment of AIDS. We synthesized new NNRTIs of the indolyl aryl sulphone (IAS) family, which are endowed with high antiviral potency against HIV-1 wt (wild-type), and the Y181C and K103N-Y181C drug resistant mutant strains. Several new compounds were highly active in lymphocytes infected with primary isolates carrying the K103N-V108I-M184V and L100I-V108I mutations. The design of new IASs was based on three-dimensional quantitative structure-activity relationship (3D QSAR) studies and docking simulations. A cross-docking study was also undertaken to gain some insights in to the binding mode of the newly synthesized IASs in the wt and mutated isoforms of reverse transcriptase

Discovery of sustainable drugs for neglected tropical diseases: cashew nutshell liquid (CNSL)-based hybrids target mitochondrial function and ATP production in Trypanosoma brucei.

In a search for effective and sustainable treatments for trypanosomiasis, we developed a library of hybrid compounds by merging the structural features of a previously synthesized quinone hit (4) with those of long‐chain phenolic constituents from cashew nut shell liquid (CNSL). CNSL is an agro‐waste product from cashew nut processing factories with great potential as a precursor for the production of drugs. The synthesized compounds were tested against Trypanosoma brucei brucei, including three multi‐drug resistant strains (B48, ISMR1, and aqp2/aqp3‐KO), T. congolense, and a human cell line (HFF). The most potent activity was found against T. b. brucei, the causative agent of African trypanosomiasis. Shorter‐chain derivatives were more active than the starting hit in parasite growth inhibition, displaying rapid trypanocidal activity with low micromolar EC50 values, but no discernable toxicity on human cell lines. Preliminary studies probing their mode of action on trypanosomes showed depletion of cellular ATP, followed by the depolarization of the mitochondrial membrane and ultrastructural damage to the mitochondrion. This was accompanied by the production of high levels of reactive oxygen species. We envisage that such hybrid compounds, obtained from renewable and inexpensive material, might be promising bio‐based, sustainable hits for anti‐trypanosomatid drug discovery

HLA-G expression and regulation during Pseudomonas aeruginosa infection in cystic fibrosis patients

Deregulated immune response fails to control biofilm-forming bacteria, as Pseudomonas aeruginosa, in the lungs of cystic fibrosis (CF) patients. HLA-G is an immune-modulatory molecule involved in respiratory diseases and infections

MudPIT analysis of released proteins in Pseudomonas aeruginosa laboratory and clinical strains in relation to pro-inflammatory effects

Pseudomonas aeruginosa (Pa) is the most common virulent pathogen contributing to the pathogenesis of cystic fibrosis (CF). During bacterial lung colonization, the products of its metabolism are released in the extracellular space contributing to the pathogenic events associated with its presence. To gain insights on the mechanisms involved in the Pa pathogenesis we focused our attention on proteins released by Pa using a MudPIT approach combined with cell biology assays. Conditioned medium (CM) collected under aerobic and microaerobic conditions from Pa clinical strains (in early and late colonization), unlike the laboratory strain, induced expression of IL-8 mRNA in CF airway epithelial cells. We have identified proteins released by clinically relevant Pa strains, focusing on the pro-inflammatory effects as metalloproteases (MMPs). In fact, their expression pattern was associated with the highest pro-inflammatory activity measured in the early clinically isolated strain. The relation was further supported by the result of the analysis of a larger and independent set of Pa isolates derived from sporadically and chronically infected CF patients: 76% of sporadic samples expressed protease activity (n = 44), while only 27% scored positive in the chronically infected individuals (n = 38, p < 0.0001, Fisher's exact test). Finally, looking for a possible mechanism of action of bacterial MMPs, we found that CM from early clinical isolates can cleave CXCR1 on the surface of human neutrophils, suggesting a potential role for the bacterially released MMPs in the protection of the pathogen from the host's response