Accelerated aging in perinatally HIV-infected children: clinical manifestations and pathogenetic mechanisms

BACKGROUND: Premature aging and related diseases have been documented in HIV-infected adults. Data are now emerging also regarding accelerated aging process in HIV-infected children. METHODS: A narrative review was performed searching studies on PubMed published in English language in 2004-2017, using appropriate key words, including "aging", "children", "HIV", "AIDS", "immunosenescence", "pathogenesis", "clinical conditions". RESULTS: Premature immunosenescence phenotype of B and T cells in HIV-infected children is mediated through immune system activation and chronic inflammation. Ongoing inflammation processes have been documented by increased levels of pathogen-associated molecular patterns (PAMPS), increased mitochondrial damage, higher levels of pro-inflammatory cytokines, and a positive correlation between sCD14 levels and percentages of activated CD8+ cells. Other reported features of premature aging include cellular replicative senescence, linked to an accelerated telomeres shortening. Finally, acceleration of age-associated methylation pattern and other epigenetic modifications have been described in HIV-infected children. All these features may favor the clinical manifestations related to premature aging. Lipid and bone metabolism, cancers, cardiovascular, renal, and neurological systems should be carefully monitored, particularly in children with detectable viremia and/or with CD4/CD8 ratio inversion. CONCLUSION: Aging processes in children with HIV infection impact their quality and length of life. Further studies regarding the mechanisms involved in premature aging are needed to search for potential targets of treatment

The rational search for selective anticancer derivatives of the peptide Trichogin GA IV: a multi-technique biophysical approach

Peptaibols are peculiar peptides produced by fungi as weapons against other microorganisms. Previous studies showed that peptaibols are promising peptide-based drugs because they act against cell membranes rather than a specific target, thus lowering the possibility of the onset of multi-drug resistance, and they possess non-coded alpha-amino acid residues that confer proteolytic resistance. Trichogin GA IV (TG) is a short peptaibol displaying antimicrobial and cytotoxic activity. In the present work, we studied thirteen TG analogues, adopting a multidisciplinary approach. We showed that the cytotoxicity is tuneable by single amino-acids substitutions. Many analogues maintain the same level of non-selective cytotoxicity of TG and three analogues are completely non-toxic. Two promising lead compounds, characterized by the introduction of a positively charged unnatural amino-acid in the hydrophobic face of the helix, selectively kill T67 cancer cells without affecting healthy cells. To explain the determinants of the cytotoxicity, we investigated the structural parameters of the peptides, their cell-binding properties, cell localization, and dynamics in the membrane, as well as the cell membrane composition. We show that, while cytotoxicity is governed by the fine balance between the amphipathicity and hydrophobicity, the selectivity depends also on the expression of negatively charged phospholipids on the cell surface

correction an epr study of ampullosporin a a medium length peptaibiotic in bicelles and vesicles

Correction for 'An EPR study of ampullosporin A, a medium-length peptaibiotic, in bicelles and vesicles' by Marco Bortolus et al., Phys. Chem. Chem. Phys., 2016, 18, 749–760

Synthesis of labeled peptides and study by EPR methods of their interaction with biological membranes

Antimicrobial peptides (AMPs) have been proposed as antitumor molecules since they sometimes displayed selective cytotoxicity towards cancer cells; they have the potential to bypass multi-drug resistance, as they exert their bioactivity by interacting with the membranes, without a specific target. Trichogin GA IV (TG) belongs to lipopeptaibols, a class of antimicrobial peptides characterized by the presence of tetrasubstituted alpha-amino acids in their sequence. Differently from other AMPs, lipopeptaibols present a significant resistance to enzyme hydrolysis. The purpose of the present Ph.D. thesis is the study of the peptide-membrane interaction in healthy and cancer cells as the membrane perturbing ability of these peptides is most likely at the basis of the selective cytotoxicity of the peptides. Working in physiological condition was of primary importance to untangle the specificity of the different peptides for the various cell lines: the membrane properties at physiological temperature are indeed among the main determinants of the peptide-membrane interaction. The study has been conducted using several techniques. Structural features of TG analogs have been quantified to rationally design new analogs with different characteristics. The results of cytotoxicity assays on cancer and healthy cells showed that it is possible to tune the toxicity of TG towards cells with different membrane composition. Fluorescence microscopy showed peptides localization on cell membranes. Electron Paramagnetic Resonance (EPR) spectroscopy is a versatile technique that has been widely exploited to determine the affinity of spin labeled peptides for model and cell membranes and their orientation in the phospholipid bilayer. The obtained results are promising, as we could relate structural modification with variations in TG analogs toxicity, an encourage further studies.I peptide antimicrobici (AMPs) sono stati proposti come possibili molecole antitumorali, poiché alcuni studi hanno evidenziato la capacità di alcuni AMPs di attaccare selettivamente alcune cellule tumorali; gli AMPs sono promettenti in quanto il loro meccanismo di azione, che prevede un attacco aspecifico sulle membrane cellulari, premette di superare lo sviluppo di resistenza ai convenzionali farmaci antibiotici ed antitumorali. La Tricogina GA IV (TG) appartiene alla famiglia dei lipopeptaibolici, una classe di peptidi antimicrobici, la cui sequenza è caratterizzata dalla presenza di alfa-ammino acidi tetrasostituiti. Diversamente da quanto osservato per altre classi di AMPs, i lipopeptaibolici sono particolarmente resistenti all’idrolisi enzimatica. Lo scopo del presente lavoro di tesi è lo studio dell’interazione peptide-membrana nel caso di cellule sane e tumorali. Infatti, la capacità di questi peptidi di perturbare le membrane è probabilmente alla base della selettività per le cellule tumorali che alcuni di essi hanno mostrato. Di primaria importanza è stato eseguire la maggior parte degli esperimenti in condizioni fisiologiche, in quanto le proprietà delle membrane a temperatura fisiologica sono tra i principali determinanti dell’interazione peptide-membrana. In questo studio sono state utilizzate diverse tecniche. Le caratteristiche strutturali degli analoghi della TG sono state quantificate per poter sviluppare un design razionale di nuovi analoghi che presentassero caratteristiche strutturali differenti. I risultati di tossicità degli analoghi della TG su cellule sane e tumorali hanno evidenziato che è in effetti possibile modulare l’attività di questi peptidi verso cellule le cui membrane presentano diversa composizione. Tramite microscopia di fluorescenza si è osservato come questi peptidi si localizzino sulle membrane cellulari. La Risonanza Paramagnetica Elettronica (EPR) è una tecnica spettroscopia versatile che è stata utilizzata per determinare quale fosse l’affinità di peptidi marcati con sonde paramagnetiche per diversi sistemi membrano-mimetici e per membrane cellulari; inoltre è stata determinata l’orientazione che tali peptidi assumono quando immersi in un doppio strato fosfolipidico. I risultati ottenuti sono promettenti ed hanno evidenziato che è possibile correlare modifiche strutturali con variazioni nella tossicità degli analoghi della TG

Synthesis of labeled peptides and study by EPR methods of their interaction with biological membranes

Antimicrobial peptides (AMPs) have been proposed as antitumor molecules since they sometimes displayed selective cytotoxicity towards cancer cells; they have the potential to bypass multi-drug resistance, as they exert their bioactivity by interacting with the membranes, without a specific target. Trichogin GA IV (TG) belongs to lipopeptaibols, a class of antimicrobial peptides characterized by the presence of tetrasubstituted alpha-amino acids in their sequence. Differently from other AMPs, lipopeptaibols present a significant resistance to enzyme hydrolysis. The purpose of the present Ph.D. thesis is the study of the peptide-membrane interaction in healthy and cancer cells as the membrane perturbing ability of these peptides is most likely at the basis of the selective cytotoxicity of the peptides. Working in physiological condition was of primary importance to untangle the specificity of the different peptides for the various cell lines: the membrane properties at physiological temperature are indeed among the main determinants of the peptide-membrane interaction. The study has been conducted using several techniques. Structural features of TG analogs have been quantified to rationally design new analogs with different characteristics. The results of cytotoxicity assays on cancer and healthy cells showed that it is possible to tune the toxicity of TG towards cells with different membrane composition. Fluorescence microscopy showed peptides localization on cell membranes. Electron Paramagnetic Resonance (EPR) spectroscopy is a versatile technique that has been widely exploited to determine the affinity of spin labeled peptides for model and cell membranes and their orientation in the phospholipid bilayer. The obtained results are promising, as we could relate structural modification with variations in TG analogs toxicity, an encourage further studies.I peptide antimicrobici (AMPs) sono stati proposti come possibili molecole antitumorali, poiché alcuni studi hanno evidenziato la capacità di alcuni AMPs di attaccare selettivamente alcune cellule tumorali; gli AMPs sono promettenti in quanto il loro meccanismo di azione, che prevede un attacco aspecifico sulle membrane cellulari, premette di superare lo sviluppo di resistenza ai convenzionali farmaci antibiotici ed antitumorali. La Tricogina GA IV (TG) appartiene alla famiglia dei lipopeptaibolici, una classe di peptidi antimicrobici, la cui sequenza è caratterizzata dalla presenza di alfa-ammino acidi tetrasostituiti. Diversamente da quanto osservato per altre classi di AMPs, i lipopeptaibolici sono particolarmente resistenti all’idrolisi enzimatica. Lo scopo del presente lavoro di tesi è lo studio dell’interazione peptide-membrana nel caso di cellule sane e tumorali. Infatti, la capacità di questi peptidi di perturbare le membrane è probabilmente alla base della selettività per le cellule tumorali che alcuni di essi hanno mostrato. Di primaria importanza è stato eseguire la maggior parte degli esperimenti in condizioni fisiologiche, in quanto le proprietà delle membrane a temperatura fisiologica sono tra i principali determinanti dell’interazione peptide-membrana. In questo studio sono state utilizzate diverse tecniche. Le caratteristiche strutturali degli analoghi della TG sono state quantificate per poter sviluppare un design razionale di nuovi analoghi che presentassero caratteristiche strutturali differenti. I risultati di tossicità degli analoghi della TG su cellule sane e tumorali hanno evidenziato che è in effetti possibile modulare l’attività di questi peptidi verso cellule le cui membrane presentano diversa composizione. Tramite microscopia di fluorescenza si è osservato come questi peptidi si localizzino sulle membrane cellulari. La Risonanza Paramagnetica Elettronica (EPR) è una tecnica spettroscopia versatile che è stata utilizzata per determinare quale fosse l’affinità di peptidi marcati con sonde paramagnetiche per diversi sistemi membrano-mimetici e per membrane cellulari; inoltre è stata determinata l’orientazione che tali peptidi assumono quando immersi in un doppio strato fosfolipidico. I risultati ottenuti sono promettenti ed hanno evidenziato che è possibile correlare modifiche strutturali con variazioni nella tossicità degli analoghi della TG

Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein Nanoparticles

Modulations of synaptic membranes play an essential role in the physiological and pathological functions of the presynaptic protein α-synuclein (αSyn). Here we used solution atomic force microscopy (AFM) and electron paramagnetic resonance (EPR) spectroscopy to investigate membrane modulations caused by αSyn. We used several lipid bilayers to explore how different lipid species may regulate αSyn–membrane interactions. We found that at a protein-to-lipid ratio of ∼1/9, αSyn perturbed lipid bilayers by generating semi-transmembrane defects that only span one leaflet. In addition, αSyn coaggregates with lipid molecules to produce ∼10 nm-sized lipoprotein nanoparticles. The obtained AFM data are consistent with the apolipoprotein characteristic of αSyn. The role of anionic lipids was elucidated by comparing results from zwitterionic and anionic lipid bilayers. Specifically, our AFM measurements showed that anionic bilayers had a larger tendency of forming bilayer defects; similarly, our EPR measurements revealed that anionic bilayers exhibited more substantial changes in lipid chain mobility and bilayer polarity. We also studied the effect of cholesterol. We found that cholesterol increased the capability of αSyn in inducing bilayer defects and altering lipid chain mobility and bilayer polarity. These data can be explained by an increase in the lipid headgroup–headgroup spacing and/or specific cholesterol−αSyn interactions. Interestingly, we found an inhibitory effect of the cone-shaped phosphatidylethanolamine lipids on αSyn-induced bilayer remodeling. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization and suppress lipid extraction. Our results of lipid-dependent membrane modulations are likely relevant to αSyn functioning

Biological Aging and Immune Senescence in Children with Perinatally Acquired HIV

Chronic HIV-infected children suffer from premature aging and aging-related diseases. Viral replication induces an ongoing inflammation process, with the release of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), the activation of the immune system, and the production of proinflammatory cytokines. Although combined highly active antiretroviral therapy (ART) has significantly modified the natural course of HIV infection, normalization of T and B cell phenotype is not completely achievable; thus, many HIV-infected children display several phenotypical alterations, including higher percentages of activated cells, that favor an accelerated telomere attrition, and higher percentages of exhausted and senescent cells. All these features ultimately lead to the clinical manifestations related to premature aging and comorbidities typically observed in older general population, including non-AIDS-related malignancies. Therefore, even under effective treatment, the premature aging process of HIV-infected children negatively impacts their quality and length of life. This review examines the available data on the impact of HIV and ART on immune and biological senescence of HIV-infected children