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Milena NASI
Professore Associato
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa
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Pubblicazioni
2024
- Vascular "Long COVID": A New Vessel Disease?
[Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Roncati, Luca; Coppi, Francesca; Nasi, Milena; Farinetti, Alberto; Manenti, Antonio; Pinti, Marcello; Mattioli, Anna Vittoria
abstract
: Vascular sequelae following (SARS-CoV-2 coronavirus disease) (COVID)-19 infection are considered as "Long Covid (LC)" disease, when occurring 12 weeks after the original infection. The paucity of specific data can be obviated by translating pathophysiological elements from the original Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection (In a microcirculatory system, a first "endotheliitis," is often followed by production of "Neutrophil Extracellular Trap," and can evolve into a more complex leukocytoklastic-like and hyperimmune vasculitis. In medium/large-sized vessels, this corresponds to endothelial dysfunction, leading to an accelerated progression of pre-existing atherosclerotic plaques through an increased deposition of platelets, circulating inflammatory cells and proteins. Associated dysregulated immune and pro-coagulant conditions can directly cause thrombo-embolic arterial or venous complications. In order to implement appropriate treatment, physicians need to consider vascular pathologies observed after SARS-Cov-2 infections as possible "LC" disease.
2023
- A Comprehensive Analysis of Cytokine Network in Centenarians
[Articolo su rivista]
Pinti, M.; Gibellini, L.; Lo Tartaro, D.; De Biasi, S.; Nasi, M.; Borella, R.; Fidanza, L.; Neroni, A.; Troiano, L.; Franceschi, C.; Cossarizza, A.
abstract
Cytokines have been investigated extensively in elderly people, with conflicting results. We performed a comprehensive analysis of the plasma levels of 62 cytokines and growth factors involved in the regulation of the immune system, in healthy centenarians, and middle-aged controls. We confirmed the previously observed increase in the levels of several pro-inflammatory cytokines, such as TNF-α and IL-6, and found that several other cytokines, directly or indirectly involved in inflammation (such as IFN-α, IL-23, CCL-5), were present at higher levels in centenarians. We did not observe any increase in the levels of anti-inflammatory cytokines, with the notable exception of the Th2-shifting cytokine IL-19. No relevant difference was observed in cytokines regulating T cell immunity. Several growth factors having a role in regulating immunity, such as G-CSF, GM-CSF, EGF, and VEGF, were upregulated in centenarians, too. Principal component analysis of the cytokine dataset showed that pro and anti-inflammatory cytokines were the variables that contributed the most to the variability of the data we observed.
2023
- Cardiovascular prevention in women: an update by the Italian Society of Cardiology working group on 'Prevention, hypertension and peripheral disease'
[Articolo su rivista]
Mattioli, Anna Vittoria; Moscucci, Federica; Sciomer, Susanna; Maffei, Silvia; Nasi, Milena; Pinti, Marcello; Bucciarelli, Valentina; Dei Cas, Alessandra; Parati, Gianfranco; Ciccone, Marco Matteo; Palmiero, Pasquale; Maiello, Maria; Pedrinelli, Roberto; Pizzi, Carmine; Barillà, Francesco; Gallina, Sabina
abstract
: The recent pandemic has substantially changed the approach to the prevention of cardiovascular diseases in women. Women have been significantly impacted by the changes that occurred during the pandemic and the quarantine adopted to prevent the spread of the disease. Changes involved prevention both through the reduction of visits and preventive screening and through social and economic changes. It is necessary to adopt new cardiovascular prevention approaches focused on returning to healthy lifestyles, reducing stress and depression also using modern tools such as telemedicine, mobile phone applications and the web. These tools convey messages in a persuasive way especially in young and adult women. There is less impact of these new tools on older women towards whom it is important to adopt a more traditional approach. This review focuses on the new approach to cardiovascular prevention in women in light of the lifestyle changes recorded during the pandemic and which led to an increase in obesity examines the effects on the cardiovascular system induced by stress and depression and analyses the new high blood pressure guidelines and indications that are specific to women.
2023
- Clodronate Liposome-Mediated Phagocytic Hemocyte Depletion Affects the Regeneration of the Cephalic Tentacle of the Invasive Snail, Pomacea canaliculata
[Articolo su rivista]
Bergamini, Giulia; Sacchi, Sandro; Ferri, Anita; Franchi, Nicola; Montanari, Monica; Ahmad, Mohamad; Losi, Chiara; Nasi, Milena; Cocchi, Marina; Malagoli, Davide
abstract
After amputation, granular hemocytes infiltrate the blastema of regenerating cephalic
tentacles of the freshwater snail Pomacea canaliculata. Here, the circulating phagocytic hemocytes were chemically depleted by injecting the snails with clodronate liposomes, and the effects on the cephalic tentacle regeneration onset and on Pc-Hemocyanin, Pc-transglutaminase (Pc-TG) and Pc-Allograft Inflammatory Factor-1 (Pc-AIF-1) gene expressions were investigated. Flow cytometry analysis demonstrated that clodronate liposomes targeted large circulating hemocytes, resulting in a transient decrease in their number. Corresponding with the phagocyte depletion, tentacle regeneration onset
was halted, and it resumed at the expected pace when clodronate liposome effects were no longer visible. In addition to the regeneration progress, the expressions of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1, which are markers of hemocyte-mediated functions like oxygen transport and immunity, clotting, and inflammation, were modified. After the injection of clodronate liposomes, a specific computer-assisted image analysis protocol still evidenced the presence of granular hemocytes in the tentacle blastema. This is consistent with reports indicating the large and agranular hemocyte population as the most represented among the professional phagocytes of P. canaliculata and with the hypothesis that different hemocyte morphologies could exert diverse biological functions, as it has been observed in other invertebrates.
2023
- Healthy preterm newborns: Altered innate immunity and impaired monocyte function
[Articolo su rivista]
De Biasi, Sara; Neroni, Anita; Nasi, Milena; Lo Tartaro, Domenico; Borella, Rebecca; Gibellini, Lara; Lucaccioni, Laura; Bertucci, Emma; Lugli, Licia; Miselli, Francesca; Bedetti, Luca; Neri, Isabella; Ferrari, Fabrizio; Facchinetti, Fabio; Berardi, Alberto; Cossarizza, Andrea
abstract
: Birth prior to 37 completed weeks of gestation is referred to as preterm (PT). Premature newborns are at increased risk of developing infections as neonatal immunity is a developing structure. Monocytes, which are key players after birth, activate inflammasomes. Investigations into the identification of innate immune profiles in premature compared to full-term infants are limited. Our research includes the investigation of monocytes and NK cells, gene expression, and plasma cytokine levels to investigate any potential differences among a cohort of 68 healthy PT and full-term infants. According to high-dimensional flow cytometry, PT infants have higher proportions of CD56+/- CD16+ NK cells and immature monocytes, and lower proportions of classical monocytes. Gene expression revealed lower proportions of inflammasome activation after in vitro monocyte stimulation and the quantification of plasma cytokine levels expressed higher concentrations of alarmin S100A8. Our findings suggest that PT newborns have altered innate immunity and monocyte functional impairment, and pro-inflammatory plasmatic profile. This may explain PT infants' increased susceptibility to infectious disease and should pave the way for novel therapeutic strategies and clinical interventions.
2023
- Long COVID: A New Challenge for Prevention of Obesity in Women
[Articolo su rivista]
Mattioli, A. V.; Coppi, F.; Nasi, M.; Pinti, M.; Gallina, S.
abstract
2023
- Mitochondrial DNA as inflammatory DAMP: a warning of an aging immune system?
[Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Lopez Domenech, Sandra; Malerba, Mara; Nasi, Milena; Mattioli, Anna Vittoria; Pinti, Marcello
abstract
Senescence of the immune system is characterized by a state of chronic, subclinical, low-grade inflammation termed 'inflammaging', with increased levels of proinflammatory cytokines, both at the tissue and systemic levels. Age-related inflammation can be mainly driven by self-molecules with immunostimulant properties, named Damage/death Associated Molecular Patterns (DAMPs), released by dead, dying, injured cells or aged cells. Mitochondria are an important source of DAMPs, including mitochondrial DNA - the small, circular, double-stranded DNA molecule found in multiple copies in the organelle. mtDNA can be sensed by at least three molecules: the Toll-like receptor 9, the NLRP3 inflammasomes, and the cyclic GMP-AMP synthase (cGAS). All these sensors can lead to the release of proinflammatory cytokines when engaged. The release of mtDNA by damaged or necrotic cells has been observed in several pathological conditions, often aggravating the course of the disease. Several lines of evidence indicate that the impairment of mtDNA quality control and of the organelle homeostasis associated with aging determines an increase in the leakage of mtDNA from the organelle to the cytosol, from the cell to the extracellular space, and into plasma. This phenomenon, mirrored by an increase in mtDNA circulating levels in elderly people, can lead to the activation of different innate immune cell types, sustaining the chronic inflammatory status that is characteristic of aging.
2023
- Physical Activity and Diet in Older Women: A Narrative Review
[Articolo su rivista]
Mattioli, Anna Vittoria; Selleri, Valentina; Zanini, Giada; Nasi, Milena; Pinti, Marcello; Stefanelli, Claudio; Fedele, Francesco; Gallina, Sabina
abstract
2023
- Reply to: Letter on the Recent Paper "Vascular 'Long COVID': A New Vessel Disease?"
[Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Roncati, Luca; Coppi, Francesca; Nasi, Milena; Farinetti, Alberto; Manenti, Antonio; Pinti, Marcello; Mattioli, Anna Vittoria
abstract
2023
- Sex and Gender Differences in Medical Education: The Impact on Scientific Reports
[Articolo su rivista]
Mattioli, A. V.; Coppi, F.; Bucciarelli, V.; Nasi, M.; Pinti, M.; Palumbo, C.; Gallina, S.
abstract
This commentary explores the reasons why sex and gender differences must be included in medical education and the impact on healthcare outcomes for patients. Understanding sex and gender differences could be useful in making more accurate diagnoses and to develop more effective treatment plans. Sex and gender medicine take into consideration both the genetic basis and the effects of exposure to environmental and socio-economic factors.
2023
- Sex Related Differences in the Complex Relationship between Coffee, Caffeine and Atrial Fibrillation
[Articolo su rivista]
Coppi, F; Bucciarelli, V; Sinigaglia, G; Zanini, G; Selleri, V; Nasi, M; Pinti, M; Gallina, S; Mattioli, Av
abstract
: This literature review aims to explore the data of articles published on the association between coffee, caffeine and atrial fibrillation and to analyze any differences between the two sexes. Several factors influence this complex relationship; genetic, environmental and psychosocial factors come into play in the pathophysiology of atrial fibrillation. These factors are expressed differently in women and men. However, the analysis of the literature has shown that comparison works between the two sexes are extremely rare. Most population-based and prospective studies either analyze aggregated data or focus on exclusively male or female populations. This results in a lack of information that could be useful in the prevention of and treatment approach to atrial fibrillation. It is necessary to deepen this issue with dedicated studies.
2023
- The Role of Lonp1 on Mitochondrial Functions during Cardiovascular and Muscular Diseases
[Articolo su rivista]
Zanini, G.; Selleri, V.; Malerba, M.; Solodka, K.; Sinigaglia, G.; Nasi, M.; Mattioli, A. V.; Pinti, M.
abstract
The mitochondrial protease Lonp1 is a multifunctional enzyme that regulates crucial mitochondrial functions, including the degradation of oxidized proteins, folding of imported proteins and maintenance the correct number of copies of mitochondrial DNA. A series of recent studies has put Lonp1 at the center of the stage in the homeostasis of cardiomyocytes and muscle skeletal cells. During heart development, Lonp1 allows the metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation. Knock out of Lonp1 arrests heart development and determines cardiomyocyte apoptosis. In adults, Lonp1 acts as a cardioprotective protein, as its upregulation mitigates cardiac injury by preventing the oxidative damage of proteins and lipids, and by preserving mitochondrial redox balance. In skeletal muscle, Lonp1 is crucial for cell development, as it mediates the activation of PINK1/Parkin pathway needed for proper myoblast differentiation. Skeletal muscle-specific ablation of Lonp1 in mice causes reduced muscle fiber size and strength due to the accumulation of mitochondrial-retained protein in muscle. Lonp1 expression and activity decline with age in different tissues, including skeletal muscle, and are associated with a functional decline and structural impairment of muscle fibers. Aerobic exercise increases unfolded protein response markers including Lonp1 in the skeletal muscle of aged animals and is associated with muscle functional recovery. Finally, mutations of Lonp1 cause a syndrome named CODAS (Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies) characterized by the impaired development of multiple organs and tissues, including myocytes. CODAS patients show hypotonia and ptosis, indicative of skeletal muscle reduced performance. Overall, this body of observations points Lonp1 as a crucial regulator of mitochondrial functions in the heart and in skeletal muscle.
2022
- Adherence to an adapted physical activity program in sedentary adults
[Articolo su rivista]
Maffei, Francesca; Nasi, Milena; Natalia Vittori, Leydi; Bragonzoni, Laura; Toselli, Stefania; Tripi, Ferdinando; Mattioli, Anna Vittoria; Maietta-Latessa, Pasqualino
abstract
Background/Objective. Physical exercise plays a key role in the prevention and prognosis of chronic diseases. Despite this evidence, a low percentage of patients regularly perform physical activity. A better understanding of the variables associated with poor adherence may help to develop some strategies to encourage people to participate in exercise interventions. This study investigated the factors influencing adherence to an Adapted Physical Activity (APA) program in subjects suffering from Acute Coronary Syndrome (ACS) clinically stable, Type 2 diabetes, or metabolic syndrome. Secondary end point was to test the efficacy of the exercise training on fitness parameters (VO2, Speed and Time) by 1 km tes
2022
- Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma
[Articolo su rivista]
Golinelli, G.; Grisendi, G.; Dall'Ora, M.; Casari, G.; Spano, C.; Talami, R.; Banchelli, F.; Prapa, M.; Chiavelli, C.; Rossignoli, F.; Candini, O.; D'Amico, R.; Nasi, M.; Cossarizza, A.; Casarini, L.; Dominici, M.
abstract
Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.
2022
- Cardiovascular Effects of Whole-Body Cryotherapy in Non-professional Athletes
[Articolo su rivista]
Coppi, Francesca; Pinti, Marcello; Selleri, Valentina; Zanini, Giada; D'Alisera, Roberta; Latessa, Pasqualino Maietta; Tripi, Ferdinando; Savino, Gustavo; Cossarizza, Andrea; Nasi, Milena; Mattioli, Anna Vittoria
abstract
2022
- Characterization of Dental Pulp Stem Cells Response to Bone Substitutes Biomaterials in Dentistry
[Articolo su rivista]
Di Tinco, R.; Consolo, U.; Pisciotta, A.; Orlandi, G.; Bertani, G.; Nasi, M.; Bertacchini, J.; Carnevale, G.
abstract
Bone substitute biomaterials (BSBs) represent a promising alternative to bone autografts, due to their biocompatibility, osteoconduction, slow resorption rates, and the ability to define and maintain volume for bone gain in dentistry. Many biomaterials are tailored to provide structural and biological support for bone regeneration, and allow the migration of bone-forming cells into the bone defect. Neural crest-derived stem cells isolated from human dental pulp (hDPSCs) represent a suitable stem cell source to study the biological effects of BSBs on osteoprogenitor cells involved in the physiological bone regenerative processes. This study aimed to evaluate how three different BSBs affect the stem cell properties, osteogenic differentiation, and inflammatory properties of hDPSCs. Our data highlight that BSBs do not alter cell proliferation and stemness markers expression, nor induce any inflammatory responses. Bone metabolism data show that hDPSCs exposed to the three BSBs distinctively secrete the factors supporting osteoblast activity and osteoclast activity. Our data indicate that (i) hDPSCs are a suitable stem cell source to study the effects of BSBs, and that (ii) the formulation of BSBs may condition the biological properties of stem cells, suggesting their versatile suitability to different dentistry applications.
2022
- Depression pandemic and cardiovascular risk in the COVID-19 era and long COVID syndrome: gender makes a difference
[Articolo su rivista]
Bucciarelli, Valentina; Nasi, Milena; Bianco, Francesco; Seferovic, Jelena; Ivkovic, Vladimir; Gallina, Sabina; Mattioli, Anna Vittoria
abstract
The ongoing COVID-19 pandemic highlighted a significant interplay between cardiovascular disease (CVD), COVID-19 related inflammatory status, and depression. Cardiovascular (CV) injury is responsible for a substantial percentage of COVID-19 deaths while COVID-19 social restrictions emerged as a non-negligible risk factor for CVD as well as a variety of mental health issues, and in particular, depression. Inflammation seems to be a shared condition between these two disorders. Gender represents a potential modifying factor both in CVD and depression, as well as in COVID-19 short- and long-term outcomes, particularly in cases involving long-term COVID complications. Results from emerging studies indicate that COVID-19 pandemic affected male and female populations in different ways. Women seem to experience less severe short-term complications but suffer worse long-term COVID complications, including depression, reduced physical activity, and deteriorating lifestyle habits, all of which may impact CV risk. Here, we summarize the current state of knowledge about the interplay between COVID-19, depression, and CV risk in women.
2022
- Differential Expression of Lonp1 Isoforms in Cancer Cells
[Articolo su rivista]
Zanini, Giada; Selleri, Valentina; De Gaetano, Anna; Gibellini, Lara; Malerba, Mara; Mattioli, Anna Vittoria; Nasi, Milena; Apostolova, Nadezda; Pinti, Marcello
abstract
Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher expression of LonP1 has been associated with higher tumour aggressiveness. Besides the full-length isoform (ISO1), we identified two other isoforms of Lonp1 in humans, resulting from alternative splicing: Isoform-2 (ISO2) lacking aa 42-105 and isoform-3 (ISO3) lacking aa 1-196. An inspection of the public database TSVdb showed that ISO1 was upregulated in lung, bladder, prostate, and breast cancer, ISO2 in all the cancers analysed (including rectum, colon, cervical, bladder, prostate, breast, head, and neck), ISO3 did not show significant changes between cancer and normal tissue. We overexpressed ISO1, ISO2, and ISO3 in SW620 cells and found that the ISO1 isoform was exclusively mitochondrial, ISO2 was present in the organelle and in the cytoplasm, and ISO3 was exclusively cytoplasmatic. The overexpression of ISO1 and, at a letter extent, of ISO2 enhanced basal, ATP-linked, and maximal respiration without altering the mitochondria number or network, mtDNA amount. or mitochondrial dynamics. A higher extracellular acidification rate was observed in ISO1 and ISO2, overexpressing cells, suggesting an increase in glycolysis. Cells overexpressing the different isoforms did not show a difference in the proliferation rate but showed a great increase in anchorage-independent growth. ISO1 and ISO2, but not ISO3, determined an upregulation of EMT-related proteins, which appeared unrelated to higher mitochondrial ROS production, nor due to the activation of the MEK ERK pathway, but rather to global metabolic reprogramming of cells.
2022
- Effective Treatment of Patients Experiencing Primary, Acute HIV Infection Decreases Exhausted/Activated CD4+ T Cells and CD8+ T Memory Stem Cells
[Articolo su rivista]
Lo Tartaro, D.; Camiro-Zuniga, A.; Nasi, M.; De Biasi, S.; Najera-Avila, M. A.; Jaramillo-Jante, M. D. R.; Gibellini, L.; Pinti, M.; Neroni, A.; Mussini, C.; Soto-Ramirez, L. E.; Calva, J. J.; Belaunzaran-Zamudio, F.; Crabtree-Ramirez, B.; Hernandez-Leon, C.; Mosqueda-Gomez, J. L.; Navarro-Alvarez, S.; Perez-Patrigeon, S.; Cossarizza, A.
abstract
Several studies have identified main changes in T- and B-lymphocyte subsets during chronic HIV infection, but few data exist on how these subsets behave during the initial phase of HIV infection. We enrolled 22 HIV-infected patients during the acute stage of infection before the initiation of antiretroviral therapy (ART). Patients had blood samples drawn previous to ART initiation (T0), and at 2 (T1) and 12 (T2) months after ART initiation. We quantified cellular HIV-DNA content in sorted naïve and effector memory CD4 T cells and identified the main subsets of T- and B-lymphocytes using an 18-parameter flow cytometry panel. We identified correlations between the patients’ clinical and immunological data using PCA. Effective HIV treatment reduces integrated HIV DNA in effector memory T cells after 12 months (T2) of ART. The main changes in CD4+ T cells occurred at T2, with a reduction of activated memory, cytolytic and activated/exhausted stem cell memory T (TSCM) cells. Changes were present among CD8+ T cells since T1, with a reduction of several activated subsets, including activated/exhausted TSCM. At T2 a reduction of plasmablasts and exhausted B cells was also observed. A negative correlation was found between the total CD4+ T-cell count and IgM-negative plasmablasts. In patients initiating ART immediately following acute/early HIV infection, the fine analysis of T- and B-cell subsets has allowed us to identify and follow main modifications due to effective treatment, and to identify significant changes in CD4+ and CD8+ T memory stem cells.
2022
- Effects of Energy Drink Acute Assumption in Gastrointestinal Tract of Rats
[Articolo su rivista]
Nasi, Milena; De Gaetano, Anna; Carnevale, Gianluca; Bertoni, Laura; Selleri, Valentina; Zanini, Giada; Pisciotta, Alessandra; Caramaschi, Stefania; Reggiani Bonetti, Luca; Farinetti, Alberto; Cossarizza, Andrea; Pinti, Marcello; Manenti, Antonio; Mattioli, Anna Vittoria
abstract
...
2022
- Evidence for mitochondrial Lonp1 expression in the nucleus
[Articolo su rivista]
Gibellini, Lara; Borella, Rebecca; De Gaetano, Anna; Zanini, Giada; Tartaro, Domenico Lo; Carnevale, Gianluca; Beretti, Francesca; Losi, Lorena; De Biasi, Sara; Nasi, Milena; Forcato, Mattia; Cossarizza, Andrea; Pinti, Marcello
abstract
The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress.
2022
- Innate immunity changes in soccer players after whole-body cryotherapy
[Articolo su rivista]
Selleri, Valentina; Mattioli, Marco; Lo Tartaro, Domenico; Paolini, Annamaria; Zanini, Giada; De Gaetano, Anna; D'Alisera, Roberta; Roli, Laura; Melegari, Alessandra; Maietta, Pasqualino; Tripi, Ferdinando; Guerra, Emanuele; Chester, Johanna; Savino, Gustavo; Trenti, Tommaso; Cossarizza, Andrea; Mattioli, Anna Vittoria; Pinti, Marcello; Nasi, Milena
abstract
Whole-body cryotherapy (WBC) consists of short exposure (up to 2-3 min) to dry air at cryogenic temperatures (up to -190 degrees C) and has recently been applied for muscle recovery after injury to reduce the inflammation process. We aimed to determine the impact of cryotherapy on immunological, hormonal, and metabolic responses in non-professional soccer players (NPSPs). Nine male NPSPs (age: 20 +/- 2 years) who trained regularly over 5 consecutive days, immediately before and after each training session, were subjected to WBC treatment (WBC-t). Blood samples were collected for the evaluation of fifty analytes including hematologic parameters, serum chemistry, and hormone profiles. Monocytes phenotyping (Mo) was performed and plasmatic markers, usually increased during inflammation [CCL2, IL-18, free mitochondrial (mt)DNA] or with anti-inflammatory effects (IL2RA, IL1RN), were quantified. After WBC-t, we observed reduced levels of ferritin, mean corpuscular hemoglobin, mean platelet volume, testosterone, and estradiol, which however remain within the normal ranges. The percentage of the total, intermediates and non-classical Mo increased, while classical Mo decreased. CXCR4 expression decreased in each Mo subset. Plasma IL18 and IL2RA levels decreased, while IL1RN only exhibited a tendency to decrease and CCL2 showed a tendency to increase. Circulating mtDNA levels were not altered following WBC-t. The differences observed in monocyte subsets after WBC-t may be attributable to their redistribution into the surrounding tissue. Moreover, the decrease of CXCR4 in Mo subpopulations could be coherent with their differentiation process. Thus, WBC through yet unknown mechanisms could promote their differentiation having a role in tissue repair.
2022
- Insegnare le differenze di genere alla facoltà di medicina: uno strumento per migliorare la sicurezza e l'efficacia della prescrizione dell’attività fisica personalizzata
[Articolo su rivista]
Pinti, Marcello; Mattioli, Anna Vittoria; Nasi, Milena; Selleri, Valentina; Palumbo, Carla
abstract
ender medicine is defined by the World Health Organization (WHO) as the study of the influence of biological differences, defined by sex, and socioeconomic and cultural differences, defined by gender, on each person's health and disease status. Since gender bias is still strongly present and entrenched in medicine, recent studies indicate how crucial the teaching of gender medicine is in the training of doctors and health professionals to create not only a positive learning environment, but also a more equitable and organized healthcare system. Therefore, the aim of the following article is to highlight the importance of systematic teaching of gender medicine to improve the safety and effectiveness of prescribing personalized physical activity and sports.
2022
- Lifestyle changes during the first and second waves of the
COVID-19 pandemic in medical college students: are there
gender-related differences?
[Articolo su rivista]
Coppi, Francesca; Nasi, Milena; Sabatini, Silvia; Bellini, Pierantonio; Generali, Luigi; Mecugni, Daniela; Farinetti, Alberto; Consolo, Ugo; Mattioli, Anna Vittoria
abstract
Background and aims: The COVID-19 pandemic has seriously affected young people. The present
study aims to explore the effects of COVID-19 on lifestyle in 500 undergraduate students both during the
acute phase of the pandemic (so-called “first wave”) and during the second spread of infections (so-called
“second wave”). Gender differences were also explored. Methods and results: During the first wave we found
weight gain in 48.6% of subjects, a switch to an unhealthy diet (43%), and an increase in the amount of food
introduced (35%). Interestingly, women showed higher intake of food in order to cope, while men privileged
higher wine consumption as a coping mechanism. We observed a sharp reduction in physical activity,
increased
sedentary behaviours and deterioration in sleep quality. Stress correlates with eating to cope (r=0.86;
p<0.001); drinking to cope (r=0.83; p<0.001). Contrary to expectations, the second wave led to a situation
similar to the first. We have detected a further deterioration in quality of sleep (67% vs 77%; p<0.01) and also
a reduction in sleeping time (68.6% vs 77.7; p<0.01). Conclusions: The long pandemic has led to unhealthy
lifestyle changes in the student population of our municipality in Northern Italy. There are gender differences
in lifestyle modifications developed during the pandemic that suggest a different response to stress. Moreover,
the persistence of pandemic-related stress due to the “second wave” has severely affected the lifestyle habits of
undergraduate student. (www.actabiomedica.it)
2022
- Mitochondrial and Endoplasmic Reticulum Alterations in a Case of Amyotrophic Lateral Sclerosis Caused by TDP-43 A382T Mutation
[Articolo su rivista]
Zanini, Giada; Selleri, Valentina; Nasi, Milena; De Gaetano, Anna; Martinelli, Ilaria; Gianferrari, Giulia; Lofaro, Francesco Demetrio; Boraldi, Federica; Mandrioli, Jessica; Pinti, Marcello
abstract
Amyotrophic lateral sclerosis is the most common form of motor neuron disease. Mutations in TARDBP, the gene encoding the RNA-binding protein TDP-43, are responsible for about 5% of familial ALS. Here we report the clinical and biological features of an ALS patients with pA382T mutation in TPD-43 protein. Disease began with right hand muscles weakness, and equally involved upper and lower motor neuron with a classic phenotype, without cognitive impairment. While a family history of neurological diseases was reported, there was no evidence of familial frontotemporal dementia. Cultured fibroblasts from the patient were characterized by profound alterations of cell proteome, which impacts particularly the mitochondrial metabolic pathways and the endoplasmic reticulum. TDP-43 levels were similar to control, healthy fibroblasts, but a higher fraction localized in mitochondria. Mitochondrial network appeared fragmented, and the organelles smaller and more spheric. In agreement with impaired proteome and morphology of mitochondria, basal cell respiration was reduced. Mitochondrial DNA levels appeared normal. However, a higher amount of mitochondrial DNA was present in the cytosol, suggesting a pronounced mitochondrial DNA misplacement which can promote a pro-inflammatory response mediating by cGAS/STING. Thus, this case report further expands the clinical and pathological phenotype of A382T mutation.
2022
- Molecular and cellular immune features of aged patients with severe COVID-19 pneumonia
[Articolo su rivista]
Lo Tartaro, D.; Neroni, A.; Paolini, A.; Borella, R.; Mattioli, M.; Fidanza, L.; Quong, A.; Petes, C.; Awong, G.; Douglas, S.; Lin, D.; Nieto, J.; Gozzi, L.; Franceschini, E.; Busani, S.; Nasi, M.; Mattioli, A. V.; Trenti, T.; Meschiari, M.; Guaraldi, G.; Girardis, M.; Mussini, C.; Gibellini, L.; Cossarizza, A.; De Biasi, S.
abstract
Aging is a major risk factor for developing severe COVID-19, but few detailed data are available concerning immunological changes after infection in aged individuals. Here we describe main immune characteristics in 31 patients with severe SARS-CoV-2 infection who were >70 years old, compared to 33 subjects <60 years of age. Differences in plasma levels of 62 cytokines, landscape of peripheral blood mononuclear cells, T cell repertoire, transcriptome of central memory CD4+ T cells, specific antibodies are reported along with features of lung macrophages. Elderly subjects have higher levels of pro-inflammatory cytokines, more circulating plasmablasts, reduced plasmatic level of anti-S and anti-RBD IgG3 antibodies, lower proportions of central memory CD4+ T cells, more immature monocytes and CD56+ pro-inflammatory monocytes, lower percentages of circulating follicular helper T cells (cTfh), antigen-specific cTfh cells with a less activated transcriptomic profile, lung resident activated macrophages that promote collagen deposition and fibrosis. Our study underlines the importance of inflammation in the response to SARS-CoV-2 and suggests that inflammaging, coupled with the inability to mount a proper anti-viral response, could exacerbate disease severity and the worst clinical outcome in old patients.
2022
- Oral health habits during COVID-19 pandemic in university medical students
[Articolo su rivista]
Bellini, Pierantonio; Sabatini, Silvia; Nasi, Milena; Dall’Olio, Lucia; Generali, Luigi; Mecugni, Daniela; Consolo, Ugo; Mattioli, Anna Vittoria
abstract
2022
- Stress and cardiovascular risk burden after the pandemic: current status and future prospects
[Articolo su rivista]
Mattioli, Anna Vittoria; Coppi, Francesca; Nasi, Milena; Gallina, Sabina
abstract
Introduction: The recent COVID-19 pandemic has induced an increase in anxiety, stress and depression in the world population, prompting a re-evaluation of these well-known risk factors on cardiovascular burden. Areas covered: This short report analyzes the impact of the pandemic on stress and depression, highlighting how the phenomenon has particularly affected women and highlights the strategies that can be undertaken after the pandemic to reduce stress and depression. We have analyzed the pandemic because it has completely changed the scenario of cardiovascular risk factors with an important increase in socio-economic stressors. Expert opinion: It is still difficult to assess the damage produced on cardiovascular risk just as it is almost impossible to predict how the overwhelming and important increase in Long-Covid Syndromes will impact the population. Strong action is needed to support critical situations and to implement social campaigns aimed at restoring healthy lifestyles. Physical activity can be an easy and inexpensive tool to help cope with stress and depression.
2022
- Teaching Gender Differences at Medical School Could Improve the Safety and Efficacy of Personalized Physical Activity Prescription
[Articolo su rivista]
Mattioli, Anna Vittoria; Nasi, Milena; Pinti, Marcello; Palumbo, Carla
abstract
2021
- Aging of immune system
[Capitolo/Saggio]
Pinti, M.; De Biasi, S.; Gibellini, L.; Lo Tartaro, D.; De Gaetano, A.; Mattioli, M.; Fidanza, L.; Nasi, M.; Cossarizza, A.
abstract
In old people, many alterations of innate and adaptive immunity have been described and viewed as deleterious, hence the term immunosenescence. Immunosenescence is a complex process involving multiple reorganizational and developmentally regulated changes, rather than a simple unidirectional decline of the whole function. Whereas innate immunity is relatively well preserved in older people, adaptive immunity is more susceptible due both to the functional decline associated with the passage of time and to antigen burden to which an individual has been exposed during lifetime. Although it is conceivable that global immune changes may lead to various diseases, it is also obvious that these changes may be needed for extended survival/longevity. This chapter summarizes recent data on the dynamic reassessment of immune changes with aging.
2021
- Hypokalemia in Patients with COVID-19
[Articolo su rivista]
Alfano, G.; Ferrari, A.; Fontana, F.; Perrone, R.; Mori, G.; Ascione, E.; Magistroni, R.; Venturi, G.; Pederzoli, S.; Margiotta, G.; Romeo, M.; Piccinini, F.; Franceschi, G.; Volpi, S.; Faltoni, M.; Ciusa, G.; Bacca, E.; Tutone, M.; Raimondi, A.; Menozzi, M.; Franceschini, E.; Cuomo, G.; Orlando, G.; Santoro, A.; Di Gaetano, M.; Puzzolante, C.; Carli, F.; Bedini, A.; Milic, J.; Meschiari, M.; Mussini, C.; Cappelli, G.; Guaraldi, G.; Borghi, V.; Burastero, G.; Corradi, L.; Di Gaetano, M.; Dolci, G.; Fantini, R.; Iadisernia, V.; Larne, D.; Pellegrino, F.; Rogati, C.; Santoro, A.; Tonelli, R.; Yaacoub, D.; Alfan, S.; Marco, B.; Pulizzi, R.; Leonelli, M.; Facchini, F.; Damiano, F.; Girardis, M.; Andreotti, A.; Biagioni, E.; Bondi, F.; Busani, S.; Chierego, G.; Scotti, M.; Cossarizza, L. S. A.; Bellinazzi, C.; Borella, R.; De Biasi, S.; De Gaetano, A.; Fidanza, L.; Gibellini, L.; Iannone, A.; Tartaro, D. L.; Mattioli, M.; Nasi, M.; Paolini, A.; Pinti, M.
abstract
Background: Patients with COVID-19 experience multiple clinical conditions that may cause electrolyte imbalances. Hypokalemia is a concerning electrolyte disorder closely associated with severe complications. This study aimed to estimate prevalence, risk factors and outcome of hypokalemia in a cohort of patients with confirmed COVID-19. Methods: A retrospective analysis was conducted on 290 non-ICU admitted patients with COVID-19 at the tertiary teaching hospital of Modena, Italy, from February 16 to April 14, 2020. Results: Hypokalemia was detected in 119 out of 290 patients (41%) during hospitalization. Mean serum potassium was 3.1 ± 0.1 meq/L. The majority of patients (90.7%) patients experienced only a mild decrease in serum potassium level (3–3.4 mEq/L). Hypokalemia was associated with hypocalcemia, which was detected in 50% of subjects. Urine potassium-to-creatinine ratio, measured in a small number of patients (n = 45; 36.1%), revealed an increase of urinary potassium excretion in most cases (95.5%). Risk factors for hypokalemia were female sex (odds ratio (OR) 2.44; 95% CI 1.36–4.37; P 0.003) and diuretic therapy (OR 1.94, 95% CI 1.08–3.48; P 0.027). Hypokalemia, adjusted for sex, age and SOFA score, was not associated with ICU transfer (OR 0.52; 95% CI 0.228–1.212; P = 0.131), in-hospital mortality (OR, 0.47; 95% CI 0.170–1.324; P = 0.154) and composite outcome of ICU transfer or in-hospital mortality (OR 0.48; 95% CI 0.222–1.047; P = 0.065) in our cohort of patients. Conclusions: Hypokalemia was a frequent disorder in subjects with COVID-19. Female sex and diuretic therapy were identified as risk factors for low serum potassium levels. Hypokalemia was unrelated to ICU transfer and death in this cohort of patients.
2021
- Letter: Does obesity affect the severity of exercise-induced muscle injury? (j obes metab syndr 2021;30:132-40)
[Articolo su rivista]
Mattioli, A. V.; Coppi, F.; Nasi, M.; Pinti, M.
abstract
2021
- Microglia activation: A role for mitochondrial DNA?
[Articolo su rivista]
Pinti, M.; Ferraro, D.; Nasi, M.
abstract
2021
- Mitochondrial DNA and exercise: Implications for health and injuries in sports
[Articolo su rivista]
Zanini, G.; De Gaetano, A.; Selleri, V.; Savino, G.; Cossarizza, A.; Pinti, M.; Mattioli, A. V.; Nasi, M.
abstract
Recently, several studies have highlighted the tight connection between mitochondria and physical activity. Mitochondrial functions are important in high-demanding metabolic activities, such as endurance sports. Moreover, regular training positively affects metabolic health by increasing mitochondrial oxidative capacity and regulating glucose metabolism. Exercise could have multiple effects, also on the mitochondrial DNA (mtDNA) and vice versa; some studies have investigated how mtDNA polymorphisms can affect the performance of general athletes and mtDNA haplogroups seem to be related to the performance of elite endurance athletes. Along with several stimuli, including pathogens, stress, trauma, and reactive oxygen species, acute and intense exercise also seem to be responsible for mtDNA release into the cytoplasm and extracellular space, leading to the activation of the innate immune response. In addition, several sports are characterized by a higher frequency of injuries, including cranial trauma, associated with neurological consequences. However, with regular exercise, circulating cell-free mtDNA levels are kept low, perhaps promoting cf-mtDNA removal, acting as a protective factor against inflammation.
2021
- Mitophagy and oxidative stress: The role of aging
[Articolo su rivista]
De Gaetano, A.; Gibellini, L.; Zanini, G.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract
Mitochondrial dysfunction is a hallmark of aging. Dysfunctional mitochondria are recognized and degraded by a selective type of macroautophagy, named mitophagy. One of the main factors contributing to aging is oxidative stress, and one of the early responses to excessive reactive oxygen species (ROS) production is the induction of mitophagy to remove damaged mitochondria. However, mitochondrial damage caused at least in part by chronic oxidative stress can accumulate, and autophagic and mitophagic pathways can become overwhelmed. The imbalance of the delicate equilibrium among mitophagy, ROS production and mitochondrial damage can start, drive, or accelerate the aging process, either in physiological aging, or in pathological age-related conditions, such as Alzheimer’s and Parkinson’s diseases. It remains to be determined which is the prime mover of this imbalance, i.e., whether it is the mitochondrial damage caused by ROS that initiates the dysregulation of mitophagy, thus activating a vicious circle that leads to the reduced ability to remove damaged mitochondria, or an alteration in the regulation of mitophagy leading to the excessive production of ROS by damaged mitochondria.
2021
- Modulation of tregs and inkt by fingolimod in multiple sclerosis patients
[Articolo su rivista]
Ferraro, D.; De Biasi, S.; Simone, A. M.; Orlandi, R.; Nasi, M.; Vitetta, F.; Pinti, M.; Fogliani, M.; Meletti, S.; Cossarizza, A.; Sola, P.
abstract
The altered numbers and functions of cells belonging to immunoregulatory cell networks such as T regulatory (Tregs) and invariant Natural Killer T (iNKT) cells have been reported in Multiple Sclerosis (MS), an immune-mediated disease. We aimed to assess the frequencies of Tregs and iNKT cells in MS patients throughout a one-year treatment with fingolimod (FTY) and to correlate immunological data with efficacy and safety data. The percentage of Tregs (defined as Live Dead-CD3 + CD4 + FoxP3 + CD25++/CD127− cells) increased steadily throughout the year, while there was no significant difference in the absolute number or percentage of iNKT cells (defined as CD3 + CD14−CD19− Vα24-Jα18 TCR+ cells). However, out of all the iNKT cells, the CD8+ iNKT and CD4−CD8− double-negative (DN) cell percentages steadily increased, while the CD4+ iNKT cell percentages decreased significantly. The mean percentage of CD8+ T cells at all time-points was lower in patients with infections throughout the study. The numbers and percentages of DN iNKT cells were more elevated, considering all time-points, in patients who presented a clinical relapse. FTY may, therefore, exert its beneficial effect in MS patients through various mechanisms, including the increase in Tregs and in iNKT subsets with immunomodulatory potential such as CD8+ iNKT cells. The occurrence of infections was associated with lower mean CD8+ cell counts during treatment with FTY.
2021
- Molecular mechanisms of mtdna-mediated inflammation
[Articolo su rivista]
De Gaetano, A.; Solodka, K.; Zanini, G.; Selleri, V.; Mattioli, A. V.; Nasi, M.; Pinti, M.
abstract
Besides their role in cell metabolism, mitochondria display many other functions. Mitochondrial DNA (mtDNA), the own genome of the organelle, plays an important role in modulating the inflammatory immune response. When released from the mitochondrion to the cytosol, mtDNA is recognized by cGAS, a cGAMP which activates a pathway leading to enhanced expression of type I interferons, and by NLRP3 inflammasome, which promotes the activation of pro-inflammatory cytokines Interleukin-1beta and Interleukin-18. Furthermore, mtDNA can be bound by Toll-like receptor 9 in the endosome and activate a pathway that ultimately leads to the expression of pro-inflammatory cytokines. mtDNA is released in the extracellular space in different forms (free DNA, protein-bound DNA fragments) either as free circulating molecules or encapsulated in extracellular vesicles. In this review, we discussed the latest findings concerning the molecular mechanisms that regulate the release of mtDNA from mitochondria, and the mechanisms that connect mtDNA misplacement to the activation of inflammation in different pathophysiological conditions.
2021
- NLRP3 and IL-1β gene expression is elevated in monocytes from HIV treated patients with neurocognitive disorders
[Articolo su rivista]
Mazaheri-Tehrani, Elham; Mohraz, Minoo; Nasi, Milena; Chester, Johanna; De Gaetano, Anna; Lo Tartaro, Domenico; Seyedalinaghi, Seyedahmad; Gholami, Mohammad; De Biasi, Sara; Gibellini, Lara; Mattioli, Anna Vittoria; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract
Systemic immune activation and inflammation in chronic HIV infection are driving factors of non-AIDS related events, including neurocognitive impairment. The role of inflammasome in monocytes from patients with HIV infection has been extensively studied but its association with the extent of neurocognitive dysfunction has been poorly investigated.
2021
- Physical Activity, Sedentary Behaviour, and Diet in Menopausal Women: Comparison Between COVID19 “first wave” and “second wave” of pandemic in Italy
[Articolo su rivista]
Coppi, Francesca; Nasi, Milena; Farinetti, Alberto; Manenti, Antonio; Gallina, Sabina; Mattioli, Anna Vittoria
abstract
The COVID-19 pandemic has strongly affected young population all over the world. The present
study explores the effects of COVID-19 on physical activity, sedentary behaviour, and diet in a population
of 435 menopausal women during the first phase of the pandemic (so-called the “first wave”) and during the
second spread of infections, after the summer release (so-called “second wave”). Women reported an increase
in perceived stress and emotional distress which led to an increase in amount of food (42%), an increase in
the consumption of snacks and junk food (48%) and to a switch to an unhealthy diet (46%) and weight gain
in 51% of subjects. Most women stopped any physical
2021
- Sex Difference in Access to Sports: A 1-Year Retrospective Study
[Articolo su rivista]
Nasi, M.; D'Alisera, R.; Cossarizza, A.; Guerra, E.; Savino, G.; Mattioli, A. V.
abstract
Purpose. Regular physical activity is a cornerstone in the prevention and treatment of cardiovascular disease thanks to its anti-inflammatory effects. Thus, favoring the access to sports is of importance for promoting well-being. The aim of the present study was to investigate how the practice of different sports is distributed among different age groups and between men and women, by taking a picture of the medical certificate request in 2017 for sports in the population of the province of Modena, Italy. Methods. We analyzed the difference in distribution of requested medical certificate from 18 874 males and 7625 females stratified for age (<18 years, 18-40 years, and >40 years) and for different sporting disciplines (athletics, football, bike, swimming, basketball, volleyball, tennis, other team sports, other individual sports, and disabled sports). Results. Men requested medical certificates more than women (more than 2.5 times). The distribution of requested certificates differs significantly (chi-square test P <.0001) at different ages and between males and females of same age. Certificate for men aged less than 18 years were 7550 and for women were 4131 and the difference increase with age. Conclusions. In order to decrease the imbalance between men and women access to sports, it is mandatory to promote a healthy lifestyle and reduce, as consequence, cardiovascular risks, mostly in women after 40 years.
2021
- THE COMPLEX RELATIONSHIP BETWEEN CAFFEINE AND ARRHYTHMIAS
[Articolo su rivista]
Mattioli, Anna Vittoria; Nasi, Milena; Toni, Silvia; Farinetti, Alberto
abstract
2020
- Cerebrospinal fluid kappa and lambda free light chains in oligoclonal band‐negative patients with suspected multiple sclerosis
[Articolo su rivista]
Ferraro, Diana; Trovati, Alice; Bedin, Roberta; Natali, Patrizia; Franciotta, Diego; Santangelo, Mario; Camera, Valentina; Vitetta, Francesca; Varani, Manuela; Trenti, Tommaso; Gastaldi, Matteo; De Biasi, Sara; Nasi, Milena; Pinti, Marcello; Meletti, Stefano; Sola, Patrizia
abstract
Cerebrospinal fluid (CSF) free light kappa chains (kappa FLC) may be a more sensitive marker of intrathecal IgG synthesis compared to oligoclonal bands (OCBs). Our aim was to retrospectively determine the additional value of the kappa and lambda index (CSF FLC/serum FLC)/(CSF albumin/serum albumin) in predicting a Multiple Sclerosis (MS) diagnosis in a group of OCB-negative patients with suspected MS.
2020
- Circulating Mitochondrial DNA and Lipopolysaccharide-Binding Protein but Not Bacterial DNA Are Increased in Acute Human Immunodeficiency Virus Infection.
[Articolo su rivista]
Nasi, Milena; Pecorini, Simone; DE BIASI, Sara; Digaetano, Margherita; Chester, JOHANNA MARY; Aramini, Beatrice; Lo Tartaro, Domenico; Pinti, Marcello; De Gaetano, Anna; Gibellini, Lara; Mattioli, Anna Vittoria; Mussini, Cristina; Cossarizza, Andrea
abstract
Microbial translocation has been suggested as a major driver of chronic immune activation HIV infection. Thus, we compared the extent of microbial translocation in patients with acute HIV infection and patients followed after CD4-guided structured treatment interruption (STI) by measuring different circulating markers: (1) lipopolysaccharide (LPS)-binding protein (LBP), (2) bacterial DNA, (3) soluble CD14 (sCD14), and (4) mitochondrial DNA (mtDNA). Bacterial DNA and sCD14 levels were similar in all groups. Patients in acute phase showed higher levels of LBP and mtDNA. In STI, we found a positive correlation between the percentage of CD8+ T cells and bacterial DNA levels. Considering all patients, LBP was positively correlated with the percentage and the absolute count of CD8+ T cells, and with mtDNA stressing the importance of mitochondrial products in sustaining chronic immune activation.
2020
- COVID 19 outbreak: impact of the quarantine-induced stress on cardiovascular disease risk burden
[Articolo su rivista]
Mattioli, Anna Vittoria; Nasi, Milena; Cocchi, Camilla; Farinetti, Alberto
abstract
“emotional eating and reduction of physical activity lead to obesity and metabolic syndrome,
both risk factors has a pivotal role in cardiovascular risk. Obesity is also associated with an
increase risk of Type 2 diabetes
2020
- COVID-19 pandemic: the effects of quarantine on cardiovascular risk
[Articolo su rivista]
Mattioli, Anna Vittoria; Ballerini Puviani, Matteo; Nasi, Milena; Farinetti, Alberto
abstract
2020
- Effects of Caffeine on Colon: A Potential Clinical Use of Coffee in Surgical Patients
[Articolo su rivista]
Mattioli, Anna Vittoria; Nasi, Milena; Farinetti, Alberto; Gelmini, Roberta
abstract
...
2020
- Effects of whole-body cryotherapy on the innate and adaptive immune response in cyclists and runners
[Articolo su rivista]
Nasi, Milena; Bianchini, Elena; Lo Tartaro, Domenico; De Biasi, Sara; Mattioli, Marco; Paolini, Annamaria; Gibellini, Lara; Pinti, Marcello; De Gaetano, Anna; D’Alisera, Roberta; Roli, Laura; Chester, Johanna; Mattioli, Anna Vittoria; Polverari, Tomassina; Maietta, Pasqualino; Tripi, Ferdinando; Stefani, Omar; Guerra, Emanuele; Savino, Gustavo; Trenti, Tommaso; Cossarizza, Andrea
abstract
2020
- Efficient T cell compartment in HIV+ patients receiving orthotopic liver transplant and immunosuppressive therapy
[Articolo su rivista]
Franceschini, Erica; De Biasi, Sara; Digaetano, Margherita; Bianchini, Elena; Lo Tartaro, Domenico; Gibellini, Lara; Menozzi, Marianna; Zona, Stefano; Tarantino, Giuseppe; Nasi, Milena; Codeluppi, Mauro; Guaraldi, Giovanni; Magistri, Paolo; Di Benedetto, Fabrizio; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract
In patients undergoing orthotopic liver transplant (OLT), immunosuppressive (IS) treatment is mandatory and infections are leading causes of morbidity/mortality. Thus, it is essential to understand the functionality of cell-mediated immunity after OLT. The aim of the study was to identify changes in T cell phenotype and polyfunctionality in HIV+ and HIV- patients treated with IS after OLT.
2020
- Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice
[Articolo su rivista]
De Gaetano, Anna; Gibellini, Lara; Bianchini, Elena; Borella, Rebecca; De Biasi, Sara; Nasi, Milena; Boraldi, Federica; Cossarizza, Andrea; Pinti, Marcello
abstract
LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations ofLONP1have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in whichLonp1was ablated. The homozygousLonp(-/-)mouse was not vital, while the heterozygousLonp1(wt/-)showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) fromLonp1(wt/-)mice showed a reduced expression ofLonp1andTfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of theLonp1gene leads to impairment of mitochondrial ultrastructure and functions in vivo.
2020
- Increased Plasma Levels of Mitochondrial DNA and Normal Inflammasome Gene Expression in Monocytes Characterize Patients With Septic Shock Due to Multidrug Resistant Bacteria
[Articolo su rivista]
Busani, Stefano; De Biasi, Sara; Nasi, Milena; Paolini, Annamaria; Venturelli, Sophie; Tosi, Martina; Girardis, Massimo; Cossarizza, Andrea
abstract
Introduction: The activity and regulation of inflammasome is receiving increasing attention in septic shock. Moreover, there is a growing body of evidence suggesting that mitochondrial DNA (mtDNA) can play a role as biomarker of disease severity and even mortality both in adults and children in critically ill setting. However, no data are available on the amount of circulating mtDNA and inflammasome gene expression in multi-drug resistant (MDR) bacteria septic shock. For this reason, the aim of this study was to determine whether plasma mtDNA levels and inflammasome gene expression in monocytes could be related to severity in patients admitted to intensive care unit (ICU) with septic shock due to MDR pathogens. Materials and Methods: Peripheral blood mononuclear cells (PBMC) and plasma were isolated from up to 20 ml of venous blood by density gradient centrifugation in patients admitted to ICU with the diagnosis of septic shock due to MDR-bacteria. Then, CD14+ monocytes were sorted, and RNA and DNA were extracted. NLRP3, PYCARD, AIM2 and NAIP expression level was analyzed by RT-PCR. Plasma circulating mtDNA levels were quantified by digital droplet PCR. Basal and outcome characteristics of the patients were collected. Age-matched healthy subjects were chosen as controls. Results: Nineteen patients with septic shock and 20 healthy subjects were enrolled in the study. A small trend toward an increased expression of inflammasome genes was observed in septic shock patients, who also displayed a marked tendency to an increased expression of IL-18 and IL-1β genes. Circulating mtDNA levels were significantly higher in septic shock patients if compared to healthy subjects, and patients who died in ICU were characterized by higher level of mtDNA if compared to those who were dismissed after 7 days. No correlations were found between mtDNA and inflammasome level and other clinical variables. Conclusion: Despite many limitations, our data suggest that in patients with septic shock caused by MDR pathogens the expression of main inflammasome genes was comparable to that of healthy patients without infection. Furthermore, our data evidence a possible role of mtDNA as a prognostic marker of severity in septic shock from MDR.
2020
- Increased plasma levels of mitochondrial DNA and pro-inflammatory cytokines in patients with progressive multiple sclerosis
[Articolo su rivista]
Nasi, M.; Bianchini, E.; De Biasi, S.; Gibellini, L.; Neroni, A.; Mattioli, Marco; Pinti, M.; Iannone, A.; Mattioli, A. V.; Simone, A. M.; Ferraro, D.; Vitetta, F.; Sola, P.; Cossarizza, A.
abstract
The role of damage-associated molecular patterns in multiple sclerosis (MS) is under investigation. Here, we studied the contribution of circulating high mobility group box protein 1 (HMGB1) and mitochondrial DNA (mtDNA) to neuroinflammation in progressive MS. We measured plasmatic mtDNA, HMGB1 and pro-inflammatory cytokines in 38 secondary progressive (SP) patients, 35 primary progressive (PP) patients and 42 controls. Free mtDNA was higher in SP than PP. Pro-inflammatory cytokines were increased in progressive patients. In PP, tumor necrosis factor-α correlated with MS Severity Score. Thus, in progressive patients, plasmatic mtDNA and pro-inflammatory cytokines likely contribute to the systemic inflammatory status.
2020
- Mediterranean Diet as a model of sustainable, resilient and healthy diet
[Articolo su rivista]
Letizia Truzzi, Maria; Ballerini Puviani, Matteo; Tripodi, Alberto; Toni, Silvia; Farinetti, Alberto; Nasi, Milena; Mattioli, Anna Vittoria
abstract
2020
- Mitochondria, oxidative stress, cancer, and aging
[Capitolo/Saggio]
Pecorini, S.; Gibellini, L.; Biasi, S. D.; Bianchini, E.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract
In human cells, the main source of reactive oxygen species (ROS) and oxidative stress are mitochondria, the organelles where oxidative phosphorylation take place. Although ROS are an inevitable by-products of respiration, they do not necessarily have detrimental effects; low doses of ROS can have beneficial effects on cells, and their production can be finely regulated in mitochondria. Increasing ROS levels and products of the oxidative stress, which occur in aging and age-related disorders, are related to progressive dysfunction of mitochondria, due to damage to mitochondrial DNA or to oxidation and damage of mitochondrial proteins, and are also present in cancer. This chapter focuses on the regulation of ROS production in mitochondria and on the mechanisms that lead to its dysregulation in aging and cancer.
2020
- Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia
[Articolo su rivista]
Nasi, Milena; De Gaetano, Anna; Bianchini, Elena; De Biasi, Sara; Gibellini, Lara; Neroni, Anita; Mattioli, Marco; Pinti, Marcello; Tartaro, Domenico Lo; Borella, Rebecca; Mattioli, Anna Vittoria; Chester, Johanna; Melegari, Alessandra; Simone, Anna Maria; Ferraro, Diana; Vitetta, Francesca; Sola, Patrizia; Cossarizza, Andrea
abstract
Microglia are the resident innate immune cells of the central nervous system and exert functions of host defence and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O2-, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.
2020
- Obesity risk during collective quarantine for the COVID-19 epidemic
[Articolo su rivista]
Mattioli, Anna Vittoria; Pinti, Marcello; Farinetti, Alberto; Nasi, Milena
abstract
In March 2020, when COVID-19 epidemics involved several countries, the WHO defined the infection as a pandemic. Government adopted measures to prevent the diffusion of infection; i.e. quarantine and isolation. One of the consequences of quarantine-induced stress is a change in lifestyle and eating habits leading to obesity. The present commentary briefly analyzes the effects of quarantine on obesity.
2020
- Peritoneal dialysis in the time of coronavirus disease 2019
[Articolo su rivista]
Alfano, Gaetano; Fontana, Francesco; Ferrari, Annachiara; Guaraldi, Giovanni; Mussini, Cristina; Magistroni, Riccardo; Cappelli, Gianni; Bacca, Erica; Bedini, Andrea; Borghi, Vanni; Burastero, Giulia; Carli, Federica; Ciusa, Giacomo; Corradi, Luca; Cuomo, Gianluca; Digaetano, Margherita; Dolci, Giovanni; Faltoni, Matteo; Fantini, Riccardo; Franceschi, Giacomo; Franceschini, Ericad; Iadisernia, Vittorio; Larnõ, Damiano; Menozzi, Marianna; Meschiari, Marianna; Milic, Jovana; Orlando, Gabriella; Pellegrino, Francesco; Raimondi, Alessandro; Rogati, Carlotta; Santoro, Antonella; Tonelli, Roberto; Tutone, Marco; Volpi, Sara; Yaacoub, Dina; Aten, G.; Marco, Ballestri; Mori, Giacomo; Girardis, Massimo; Andreotti, Alberto; Biagioni, Emanuela; Bondi, Filippo; Busani, Stefano; Chierego, Giovanni; Scotti, Marzia; Serio, Lucia; Cossarizza, Andrea; Bellinazzi, Caterina; Borella, Rebecca; de Biasi, Sara; de Gaetano, Anna; Fidanza, Lucia; Gibellini, Lara; Iannone, Anna; Lo Tartaro, Domenico; Mattioli, Marco; Nasi, Milena; Paolini, Annamariag; Pinti, Marcello
abstract
In the current setting of global containment, peritoneal dialysis (PD) and home haemodialysis are the best modalities of renal replacement therapy (RRT) to reduce the rate of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the shorter and easier training programme of PD compared to home haemodialysis, PD appears a practical solution for patients with end-stage renal disease to reduce the risk of hospital-acquired infection. PD offers the advantage of minimizing the risk of viral transmission through interpersonal contact that commonly occurs during the haemodialysis session and while travelling from home to the haemodialysis facility using public transport services. To overcome barriers to health care access due to the containment measures for this emerging disease, telemedicine is a useful and reliable tool for delivering health care without exposing patients to the risk of contact. However, novel issues including handling of potentially infected dialysate, caregivers' infectious risk and adequacy of PD in critically ill patients with acute respiratory distress syndrome remain to be clarified. In conclusion, PD should be preferred to the other modalities of RRT during the coronavirus disease 2019 (COVID-19) outbreak because it can be a solution to cope with the increased number of infected patients worldwide.
2020
- Plasma neurofilaments correlate with disability in progressive multiple sclerosis patients
[Articolo su rivista]
Ferraro, Diana; Guicciardi, Claudio; De Biasi, Sara; Pinti, Marcello; Bedin, Roberta; Camera, Valentina; Vitetta, Francesca; Nasi, Milena; Meletti, Stefano; Sola, Patrizia
abstract
Cerebrospinal fluid (CSF) and blood neurofilaments (NFLs) are markers of axonal damage and are being investigated, mostly in relapsing-remitting (RR) MS, as a marker of disease activity and of response to treatment, while there are less data in progressive MS patients. Primary aim was to measure NFL in plasma samples of untreated patients with primary (PP) and secondary (SP) progressive MS and to correlate them with disability, disease severity, and prior/subsequent disability progression.
2020
- Relationship between socioeconomic status and asymptomatic peripheral arterial disease: a retrospective study
[Articolo su rivista]
Mattioli, Anna V; Nasi, Milena; Coppi, Francesca; Gelmini, Roberta; Farinetti, Alberto
abstract
2020
- The immune response of the invasive golden apple snail to a nematode‐based molluscicide involves different organs
[Articolo su rivista]
Montanari, Alice; Bergamini, Giulia; Ferrari, Agnese; Ferri, Anita; Nasi, Milena; Simonini, Roberto; Malagoli, Davide
abstract
Simple SummarySustainable solutions to the spreading of invasive species are difficult to find due to the absence of biological information about basic immune mechanisms of the target pests. Here, we present evidence of the effects of a commercially available roundworm, Phasmarhabditis hermaphrodita, against the invasive apple snail Pomacea canaliculata. The effects are principally evaluated in terms of snail survival and immune activation. Via molecular and microscopy-based approaches, we demonstrate that dosage and temperature are critical in determining the effects of the roundworm, and that the apple snail response to this immune challenge involves different organs. To our knowledge, these findings are the first demonstration that a P. hermaphrodita-based molluscicide can effectively kill P. canaliculata and that the snail can mount a multi-organ response against this pathogenic roundworm.The spreading of alien and invasive species poses new challenges for the ecosystem services, the sustainable production of food, and human well-being. Unveiling and targeting the immune system of invasive species can prove helpful for basic and applied research. Here, we present evidence that a nematode (Phasmarhabditis hermaphrodita)-based molluscicide exerts dose-dependent lethal effects on the golden apple snail, Pomacea canaliculata. When used at 1.7 g/L, this biopesticide kills about 30% of snails within one week and promotes a change in the expression of Pc-bpi, an orthologue of mammalian bactericidal/permeability increasing protein (BPI). Changes in Pc-bpi expression, as monitored by quantitative PCR (qPCR), occurred in two immune-related organs, namely the anterior kidney and the gills, after exposure at 18 and 25 degrees C, respectively. Histological analyses revealed the presence of the nematode in the snail anterior kidney and the gills at both 18 and 25 degrees C. The mantle and the central nervous system had a stable Pc-bpi expression and seemed not affected by the nematodes. Fluorescence in situ hybridization (FISH) experiments demonstrated the expression of Pc-bpi in circulating hemocytes, nurturing the possibility that increased Pc-bpi expression in the anterior kidney and gills may be due to the hemocytes patrolling the organs. While suggesting that P. hermaphrodita-based biopesticides enable the sustainable control of P. canaliculata spread, our experiments also unveiled an organ-specific and temperature-dependent response in the snails exposed to the nematodes. Overall, our data indicate that, after exposure to a pathogen, the snail P. canaliculata can mount a complex, multi-organ innate immune response.
2020
- The importance of advanced cytometry in defining new immune cell types and functions relevant for the immunopathogenesis of HIV infection
[Articolo su rivista]
Agrati, C.; de Biasi, S.; Fidanza, L.; Gibellini, L.; Nasi, M.; Pinti, M.; Cossarizza, A.
abstract
In the last years, novel, exciting immunological findings of interest for HIV research and treatment were identified thanks to different cytometric approaches. The analysis of the phenotypes and functionality of cells belonging to the immune system could clarify their role in the immunopathogenesis of HIV infection, and to elaborate key concepts, relevant in the treatment of this disease. Important discoveries have been made concerning cells that are important for protective immunity like lymphocytes that display polyfunctionality, resident memory T cells, innate lymphoid cells, to mention a few. The complex phenotype of myeloid-derived suppressor cells has been investigated, and relevant changes have been reported during chronic and primary HIV infection, in correlation with changes in CD4þ T-cell number, T-cell activation, and with advanced disease stage. The search for markers of HIV persistence present in latently infected cells, namely those molecules that are important for a functional or sterilizing cure, evidenced the role of follicular helper T cells, and opened a discussion on the meaning and use of different surface molecules not only in identifying such cells, but also in designing new strategies. Finally, advanced technologies based upon the simultaneous detection of HIV-RNA and proteins at the single cell level, as well as those based upon spectral cytometry or mass cytometry are now finding new actors and depicting a new scenario in the immunopathogenesis of the infection, that will allow to better design innovative therapies based upon novel drugs and vaccines.
2019
- Altered Expression of PYCARD, Interleukin 1β, Interleukin 18, and NAIP in Successfully Treated HIV-Positive Patients With a Low Ratio of CD4+ to CD8+ T Cells
[Articolo su rivista]
Nasi, Milena; Pecorini, Simone; De Biasi, Sara; Bianchini, Elena; Digaetano, Margherita; Neroni, Anita; Lo Tartaro, Domenico; Pullano, Rosalberta; Pinti, Marcello; Gibellini, Lara; Mussini, Cristina; Cossarizza, Andrea
abstract
The expression and activity of main inflammasome components in monocytes from successfully treated HIV+ patients are poorly studied. Thus, we enrolled 18 patients with low and 17 with normal CD4/CD8 ratio compared to 11 healthy donors. Our results show that patients with low ratio have a decreased CCR2 expression among classical and intermediate monocytes and an increased CCR5 expression among classical, compared to whose with normal ratio. They also showed higher NAIP and PYCARD mRNA levels after LPS-stimulation suggesting an altered ability to control immune activation that could affect their immune reconstitution.
2019
- Effects of a nematode-based molluscicide on survival and antimicrobial peptide expression in Pomacea canaliculata
[Abstract in Atti di Convegno]
Montanari, A; Accorsi, A; Nasi, M; Malagoli, D
abstract
2019
- Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)
[Articolo su rivista]
Cossarizza, A.; Chang, H. -D.; Radbruch, A.; Acs, A.; Adam, D.; Adam-Klages, S.; Agace, W. W.; Aghaeepour, N.; Akdis, M.; Allez, M.; Almeida, L. N.; Alvisi, G.; Anderson, G.; Andra, I.; Annunziato, F.; Anselmo, A.; Bacher, P.; Baldari, C. T.; Bari, S.; Barnaba, V.; Barros-Martins, J.; Battistini, L.; Bauer, W.; Baumgart, S.; Baumgarth, N.; Baumjohann, D.; Baying, B.; Bebawy, M.; Becher, B.; Beisker, W.; Benes, V.; Beyaert, R.; Blanco, A.; Boardman, D. A.; Bogdan, C.; Borger, J. G.; Borsellino, G.; Boulais, P. E.; Bradford, J. A.; Brenner, D.; Brinkman, R. R.; Brooks, A. E. S.; Busch, D. H.; Buscher, M.; Bushnell, T. P.; Calzetti, F.; Cameron, G.; Cammarata, I.; Cao, X.; Cardell, S. L.; Casola, S.; Cassatella, M. A.; Cavani, A.; Celada, A.; Chatenoud, L.; Chattopadhyay, P. K.; Chow, S.; Christakou, E.; Cicin-Sain, L.; Clerici, M.; Colombo, F. S.; Cook, L.; Cooke, A.; Cooper, A. M.; Corbett, A. J.; Cosma, A.; Cosmi, L.; Coulie, P. G.; Cumano, A.; Cvetkovic, L.; Dang, V. D.; Dang-Heine, C.; Davey, M. S.; Davies, D.; De Biasi, S.; Del Zotto, G.; Dela Cruz, G. V.; Delacher, M.; Della Bella, S.; Dellabona, P.; Deniz, G.; Dessing, M.; Di Santo, J. P.; Diefenbach, A.; Dieli, F.; Dolf, A.; Dorner, T.; Dress, R. J.; Dudziak, D.; Dustin, M.; Dutertre, C. -A.; Ebner, F.; Eckle, S. B. G.; Edinger, M.; Eede, P.; Ehrhardt, G. R. A.; Eich, M.; Engel, P.; Engelhardt, B.; Erdei, A.; Esser, C.; Everts, B.; Evrard, M.; Falk, C. S.; Fehniger, T. A.; Felipo-Benavent, M.; Ferry, H.; Feuerer, M.; Filby, A.; Filkor, K.; Fillatreau, S.; Follo, M.; Forster, I.; Foster, J.; Foulds, G. A.; Frehse, B.; Frenette, P. S.; Frischbutter, S.; Fritzsche, W.; Galbraith, D. W.; Gangaev, A.; Garbi, N.; Gaudilliere, B.; Gazzinelli, R. T.; Geginat, J.; Gerner, W.; Gherardin, N. A.; Ghoreschi, K.; Gibellini, L.; Ginhoux, F.; Goda, K.; Godfrey, D. I.; Goettlinger, C.; Gonzalez-Navajas, J. M.; Goodyear, C. S.; Gori, A.; Grogan, J. L.; Grummitt, D.; Grutzkau, A.; Haftmann, C.; Hahn, J.; Hammad, H.; Hammerling, G.; Hansmann, L.; Hansson, G.; Harpur, C. M.; Hartmann, S.; Hauser, A.; Hauser, A. E.; Haviland, D. L.; Hedley, D.; Hernandez, D. C.; Herrera, G.; Herrmann, M.; Hess, C.; Hofer, T.; Hoffmann, P.; Hogquist, K.; Holland, T.; Hollt, T.; Holmdahl, R.; Hombrink, P.; Houston, J. P.; Hoyer, B. F.; Huang, B.; Huang, F. -P.; Huber, J. E.; Huehn, J.; Hundemer, M.; Hunter, C. A.; Hwang, W. Y. K.; Iannone, A.; Ingelfinger, F.; Ivison, S. M.; Jack, H. -M.; Jani, P. K.; Javega, B.; Jonjic, S.; Kaiser, T.; Kalina, T.; Kamradt, T.; Kaufmann, S. H. E.; Keller, B.; Ketelaars, S. L. C.; Khalilnezhad, A.; Khan, S.; Kisielow, J.; Klenerman, P.; Knopf, J.; Koay, H. -F.; Kobow, K.; Kolls, J. K.; Kong, W. T.; Kopf, M.; Korn, T.; Kriegsmann, K.; Kristyanto, H.; Kroneis, T.; Krueger, A.; Kuhne, J.; Kukat, C.; Kunkel, D.; Kunze-Schumacher, H.; Kurosaki, T.; Kurts, C.; Kvistborg, P.; Kwok, I.; Landry, J.; Lantz, O.; Lanuti, P.; Larosa, F.; Lehuen, A.; LeibundGut-Landmann, S.; Leipold, M. D.; Leung, L. Y. T.; Levings, M. K.; Lino, A. C.; Liotta, F.; Litwin, V.; Liu, Y.; Ljunggren, H. -G.; Lohoff, M.; Lombardi, G.; Lopez, L.; Lopez-Botet, M.; Lovett-Racke, A. E.; Lubberts, E.; Luche, H.; Ludewig, B.; Lugli, E.; Lunemann, S.; Maecker, H. T.; Maggi, L.; Maguire, O.; Mair, F.; Mair, K. H.; Mantovani, A.; Manz, R. A.; Marshall, A. J.; Martinez-Romero, A.; Martrus, G.; Marventano, I.; Maslinski, W.; Matarese, G.; Mattioli, A. V.; Maueroder, C.; Mazzoni, A.; Mccluskey, J.; Mcgrath, M.; Mcguire, H. M.; Mcinnes, I. B.; Mei, H. E.; Melchers, F.; Melzer, S.; Mielenz, D.; Miller, S. D.; Mills, K. H. G.; Minderman, H.; Mjosberg, J.; Moore, J.; Moran, B.; Moretta, L.; Mosmann, T. R.; Muller, S.; Multhoff, G.; Munoz, L. E.; Munz, C.; Nakayama, T.; Nasi, M.; Neumann, K.; Ng, L. G.; Niedobitek, A.; Nourshargh, S.; Nunez, G.; O'Connor, J. -E.; Ochel, A.; Oja, A.; Ordonez, D.; Orfao, A.; Orlowski-Oliver, E.; Ouyang, W.; Oxenius, A.; Palankar, R.; Panse, I.; Pattanapanyasat, K.; Paulsen, M.; Pavlinic, D.; Penter,
abstract
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
2019
- Mitochondrial functionality and metabolism in T cells from progressive multiple sclerosis patients
[Articolo su rivista]
De Biasi, Sara; Simone, Anna Maria; Bianchini, Elena; Lo Tartaro, Domenico; Pecorini, Simone; Nasi, Milena; Patergnani, Simone; Carnevale, Gianluca; Gibellini, Lara; Ferraro, Diana; Vitetta, Francesca; Pinton, Paolo; Sola, Patrizia; Cossarizza, Andrea; Pinti, Marcello
abstract
Patients with primary progressive (PP) and secondary progressive (SP) forms of multiple sclerosis (MS) exhibit a sustained increase in the number of Th1, T cytotoxic type-1 and Th17 cells in peripheral blood, suggesting that the progressive phase is characterized by a permanent peripheral immune activation. As T cell functionality and activation are strictly connected to their metabolic profile, we investigated the mitochondrial functionality and metabolic changes of T cell subpopulations in a cohort of progressive MS patients. T cells from progressive patients were characterized by low proliferation and increase of terminally differentiated/exhausted cells. T cells from PP patients showed lower Oxygen Consumption Rate and Extracellular Acidification Rate, lower mitochondrial mass, membrane potential and respiration than those of SP patients, a downregulation of transcription factors supporting respiration and higher tendency to shift towards glycolysis upon stimulation. Furthermore, PP effector memory T cells were characterized by higher Glucose transporter -1 levels and a higher expression of glycolytic-supporting genes if compared to SP patients. Overall, our data suggest that profound differences exist in the phenotypic and metabolic features of T cells from PP and SP patients, even though the two clinical phenotypes are considered part of the same disease spectrum.
2019
- Sporadic and hereditary hemangioblastoma: The role of endothelial cells
[Articolo su rivista]
Feletti, A.; Bianchini, E.; De Gaetano, A.; Gibellini, L.; De Biasi, S.; Pavesi, G.; Mattioli, A. V.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract
2019
- Synthesis and anticancer activity of CDDO and CDDO-me, two derivatives of natural triterpenoids
[Articolo su rivista]
Borella, R.; Forti, L.; Gibellini, L.; De Gaetano, A.; De Biasi, S.; Nasi, M.; Cossarizza, A.; Pinti, M.
abstract
Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me.
2019
- The role of physical activity in individuals with cardiovascular risk factors: an opinion paper from Italian Society of Cardiology-Emilia Romagna-Marche and SIC-Sport
[Articolo su rivista]
Nasi, Milena; Patrizi, Giampiero; Pizzi, Carmine; Landolfo, Matteo; Boriani, Giuseppe; Dei Cas, Alessandra; Cicero, Arrigo F G; Fogacci, Federica; Rapezzi, Claudio; Sisca, Giovanbattista; Capucci, Alessandro; Vitolo, Marco; Galiè, Nazzareno; Borghi, Claudio; Berrettini, Umberto; Piepoli, Massimo; Mattioli, Anna V
abstract
: Regular physical activity is a cornerstone in the prevention and treatment of atherosclerotic cardiovascular disease (CVD) due to its positive effects in reducing several cardiovascular risk factors. Current guidelines on CVD suggest for healthy adults to perform at least 150 min/week of moderate intensity or 75 min/week of vigorous intensity aerobic physical activity. The current review explores the effects of physical activity on some risk factors, specifically: diabetes, dyslipidemia, hypertension and hyperuricemia. Physical activity induces an improvement in insulin sensitivity and in glucose control independently of weight loss, which may further contribute to ameliorate both diabetes-associated defects. The benefits of adherence to physical activity have recently proven to extend beyond surrogate markers of metabolic syndrome and diabetes by reducing hard endpoints such as mortality. In recent years, obesity has greatly increased in all countries. Weight losses in these patients have been associated with improvements in many cardiometabolic risk factors. Strategies against obesity included caloric restriction, however greater results have been obtained with association of diet and physical activity. Similarly, the beneficial effect of training on blood pressure via its action on sympathetic activity and on other factors such as improvement of endothelial function and reduction of oxidative stress can have played a role in preventing hypertension development in active subjects. The main international guidelines on prevention of CVD suggest to encourage and to increase physical activity to improve lipid pattern, hypertension and others cardiovascular risk factor. An active action is required to the National Society of Cardiology together with the Italian Society of Sports Cardiology to improve the prescription of organized physical activity in patients with CVD and/or cardiovascular risk factors.
2019
- Titanium Surface Properties Influence the Biological Activity and FasL Expression of Craniofacial Stromal Cells.
[Articolo su rivista]
Conserva, E; Pisciotta, A; Borghi, F; Nasi, M; Pecorini, Simone; Bertoni, L; de Pol, A; Consolo, U; Carnevale, G.
abstract
Mesenchymal stromal cells (MSCs) can be easily isolated form craniofacial bones during routine dentistry procedures. Due to their embryological origin from neural crest, they represent a suitable cell population to study cell-biomaterial interaction in the craniofacial field, including osteoinductive/osteointegrative processes. The biological and immunomodulatory properties of MSCs may be influenced by chemistry and topography of implant surfaces. We investigated if and how three different titanium surfaces, machined (MCH), sandblasted with resorbable blasting medium (RBM), and Ca++-nanostructured (NCA), may affect biological activity, osseointegration, and immunomodulatory properties of craniofacial MSCs. Cell proliferation, morphology, osteogenic markers, and FasL were evaluated on MSCs isolated from the mandibular bone after seeding on these three different surfaces. No statistically significant differences in cell proliferation were observed whereas different morphologies and growth patterns were detected for each type of surface. No difference in the expression of osteogenic markers was revealed. Interestingly, FasL expression, involved in the immunomodulatory activity of stem cells, was influenced by surface properties. Particularly, immunofluorescence analysis indicated that FasL expression increased on MCH surface compared to the others confirming the suggested role of FasL in promoting osteogenic differentiation. Titanium surface treatments and topography might reflect different biological behaviours of craniofacial MSCs and influence their osseointegration/immunomodulation properties.
2018
- Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR
[Articolo su rivista]
Gibellini, Lara; Pecorini, Simone; De Biasi, Sara; Pinti, Marcello; Bianchini, Elena; DE GAETANO, Anna; Digaetano, Margherita; Pullano, Rosalberta; Lo Tartaro, Domenico; Iannone, Anna; Mussini, Cristina; Cossarizza, Andrea; Nasi, Milena
abstract
Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.
2018
- High speed flow cytometry allows the detection of circulating endothelial cells in hemangioblastoma patients
[Articolo su rivista]
De Biasi, Sara; Gibellini, Lara; Feletti, Alberto; Pavesi, Giacomo; Bianchini, Elena; Lo Tartaro, Domenico; Pecorini, Simone; De Gaetano, Anna; Pullano, Rosalberta; Nasi, Milena; Pinti, Marcello; Cossarizza, Andrea; Boraldi, Federica
abstract
Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB). HB is a rare benign tumor, responsible for 1-2.5% of primary intracranial tumors and up to 10% of spinal cord tumors, and for which no tools are available to predict the onset or recurrence in patients undergoing surgical removal of tumor mass. This method allowed us to accurately quantifying CEC and CEP before and after surgery. CEPs are present at high levels in HB patients than control before intervention, and decrease after tumor removal, suggesting that their percentage could represent a valid tool to monitor the disease onset and recurrence.
2018
- LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells
[Articolo su rivista]
Gibellini, L; Losi, L; De Biasi, S; Nasi, M; Lo Tartaro, D; Pecorini, S; Patergnani, S; Pinton, P; De Gaetano, A; Carnevale, G; Pisciotta, A; Mariani, F; Roncucci, L; Iannone, A; Cossarizza, A; Pinti, M.
abstract
Mitochondrial Lon protease (LonP1) is a multi-function enzyme that regulates mitochondrial functions in several human malignancies, including colorectal cancer (CRC). The mechanism(s) by which LonP1 contributes to colorectal carcinogenesis is not fully understood. We found that silencing LonP1 leads to severe mitochondrial impairment and apoptosis in colon cancer cells. Here, we investigate the role of LonP1 in mitochondrial functions, metabolism, and epithelial-mesenchymal transition (EMT) in colon tumor cells and in metastasis. LonP1 was almost absent in normal mucosa, gradually increased from aberrant crypt foci to adenoma, and was most abundant in CRC. Moreover, LonP1 was preferentially upregulated in colorectal samples with mutated p53 or nuclear β-catenin, and its overexpression led to increased levels of β-catenin and decreased levels of E-cadherin, key proteins in EMT, in vitro. LonP1 upregulation also induced opposite changes in oxidative phosphorylation, glycolysis, and pentose pathway in SW480 primary colon tumor cells when compared to SW620 metastatic colon cancer cells. In conclusion, basal LonP1 expression is essential for normal mitochondrial function, and increased LonP1 levels in SW480 and SW620 cells induce a metabolic shift toward glycolysis, leading to EMT.
2018
- Real time PCR and image-based cell profiling analyses evidence correlation between hematopoietic and ampulla cell populations in the gastropod Pomacea canaliculata
[Abstract in Atti di Convegno]
Accorsi, A; Montanari, A; Nasi, M; Pecorini, S; Box, A; Peuß, R; Malagoli, D
abstract
2018
- The role of latency reversal agents in the cure of HIV: A review of current data
[Articolo su rivista]
Bashiri, Kiandokht; Rezaei, Nima; Nasi, Milena; Cossarizza, Andrea
abstract
The definitive cure for human immunodeficiency virus type-1 (HIV) infection is represented by the eradication of the virus from the patient's body. To reach this result, cells that are infected but do not produce the virus must become recognizable to be killed by the immune system. For this purpose, drugs defined "latency reverting agents" (LRA) that reactivate viral production are under investigation. A few clinical studies have been performed in HIV-infected patients treated with LRA and combined antiretroviral therapy (cART). The strategy is thus to combine cART and LRA to reactivate the virus and unmask latently infected cells that, because of cART, cannot produce a fully competent form of the virus. Unmasked cells can present viral antigens to the immune system, that ultimately recognizes and kills such latently infected cells. This review reports and discusses recent studies that have been published on this topic.
2017
- A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata
[Articolo su rivista]
Accorsi, Alice; Benatti, Stefania; Ross, Eric; Nasi, Milena; Malagoli, Davide
abstract
The golden apple snail Pomacea canaliculata is an invasive pest originating from South America. It has already been found in Asia, the southern United States and more recently in the EU. Aiming to target the immune system of the snail as a way to control its spreading, we have developed organ-specific transcriptomes and looked for molecules controlling replication and differentiation of snail hemocytes. The prokineticin domain-containing protein Astakine 1 is the only cytokine known thus far capable of regulating invertebrate hematopoiesis, and we analyzed the transcriptomes looking for molecules containing a prokineticin domain. We have identified a prokineticin-like protein (PlP), that we called Pc-plp and we analyzed by real-time PCR (qPCR) its expression. In control snails, highest levels of Pc-plp were detected in the digestive gland, the ampulla (i.e., a hemocyte reservoir) and the pericardial fluid (i.e., the hematopoietic district). We tested Pc-plp expression after triggering hematopoiesis via multiple hemolymph withdrawals, or during bacterial challenge through LPS injection. In both cases a reduction of Pc-plp mRNA was observed. The multiple hemolymph withdrawals caused a significant decrease of Pc-plp mRNA in pericardial fluid and circulating hemocytes, while the LPS injection promoted the Pc-plp mRNA drop in anterior kidney, mantle and gills, organs that may act as immune barrier in molluscs. Our data indicate an important role for prokineticin domain-containing proteins as immunomodulators also in gastropods and their dynamic expression may serve as a biosensor to gauge the effectiveness of immunological interventions aimed at curtailing the spreading of the gastropod pest P. canaliculata.
2017
- Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function
[Articolo su rivista]
Morelli F., F.; Verbeek D., S.; Bertacchini, Jessika; Vinet, Jonathan; Mediani, Laura; Marmiroli, Sandra; Cenacchi, G.; Nasi, Milena; DE BIASI, Sara; Brunsting J., F.; Lammerding, J.; Pegoraro, E.; Angelini, C.; Tupler, Rossella; Alberti, S.; Carra, Serena
abstract
Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy.
2017
- Ageing and inflammation in patients with HIV infection
[Articolo su rivista]
Nasi, Milena; DE BIASI, Sara; Gibellini, Lara; Bianchini, Elena; Pecorini, S.; Bacca, V.; Guaraldi, Giovanni; Mussini, Cristina; Pinti, Marcello; Cossarizza, Andrea
abstract
Nowadays, HIV+ patients have an expected lifespan that is only slightly shorter than healthy individuals. For this reason, along with the fact that infection can be acquired at a relatively advanced age, the effects of ageing on HIV+ people have begun to be evident. Successful anti-viral treatment is, on one hand, responsible for the development of side effects related to drug toxicity; on the other hand, it is not able to inhibit the onset of several complications caused by persistent immune activation and chronic inflammation. Therefore, patients with a relatively advanced age, i.e. aged more than 50 years, can experience pathologies that affect much older citizens. HIV+ individuals with non-AIDS-related complications can thus come to the attention of clinicians because of the presence of neurocognitive disorders, cardiovascular diseases, metabolic syndrome, bone abnormalities and non-HIV-associated cancers. Chronic inflammation and immune activation, observed typically in elderly people and defined as 'inflammaging', can be present in HIV+ patients who experience a type of premature ageing, which affects the quality of life significantly. This relatively new condition is extremely complex, and important factors have been identified as well as the traditional behavioural risk factors, e.g. the toxicity of anti-retroviral treatments and the above-mentioned chronic inflammation leading to a functional decline and a vulnerability to injury or pathologies. Here, we discuss the role of inflammation and immune activation on the most important non-AIDS-related complications of chronic HIV infection, and the contribution of aging per se to this scenario.
2017
- Basic science and pathogenesis of ageing with HIV: Potential mechanisms and biomarkers
[Articolo su rivista]
Lagathu, Claire; Cossarizza, Andrea; Bã©rã©ziat, Vã©ronique; Nasi, Milena; Capeau, Jacqueline; Pinti, Marcello
abstract
The increased prevalence of age-related comorbidities and mortality is worrisome in ageing HIV-infected patients. Here, we aim to analyse the different ageing mechanisms with regard to HIV infection. Ageing results from the time-dependent accumulation of random cellular damage. Epigenetic modifications and mitochondrial DNA haplogroups modulate ageing. In antiretroviral treatment-controlled patients, epigenetic clock appears to be advanced, and some haplogroups are associated with HIV infection severity. Telomere shortening is enhanced in HIV-infected patients because of HIV and some nucleoside analogue reverse transcriptase inhibitors. Mitochondria-related oxidative stress and mitochondrial DNA mutations are increased during ageing and also by some nucleoside analogue reverse transcriptase inhibitors. Overall, increased inflammation or 'inflammageing' is a major driver of ageing and could result from cell senescence with secreted proinflammatory mediators, altered gut microbiota, and coinfections. In HIV-infected patients, the level of inflammation and innate immunity activation is enhanced and related to most comorbidities and to mortality. This status could result, in addition to age, from the virus itself or viral protein released from reservoirs, from HIV-enhanced gut permeability and dysbiosis, from antiretroviral treatment, from frequent cytomegalovirus and hepatitis C virus coinfections, and also from personal and environmental factors, as central fat accumulation or smoking. Adaptive immune activation and immunosenescence are associated with comorbidities and mortality in the general population but are less predictive in HIV-infected patients. Biomarkers to evaluate ageing in HIV-infected patients are required. Numerous systemic or cellular inflammatory, immune activation, oxidative stress, or senescence markers can be tested in serum or peripheral blood mononuclear cells. The novel European Study to Establish Biomarkers of Human Ageing MARK-AGE algorithm, evaluating the biological age, is currently assessed in HIV-infected patients and reveals an advanced biological age. Some enhanced inflammatory or innate immune activation markers are interesting but still not validated for the patient's follow-up. To be able to assess patients' biological age is an important objective to improve their healthspan.
2017
- Guidelines for the use of flow cytometry and cell sorting in immunological studies
[Articolo su rivista]
Cossarizza, Andrea; Chang, Hyun-dong; Radbruch, Andreas; Andrã¤, Immanuel; Annunziato, Francesco; Bacher, Petra; Barnaba, Vincenzo; Battistini, Luca; Bauer, Wolfgang M.; Baumgart, Sabine; Becher, Burkhard; Beisker, Wolfgang; Berek, Claudia; Blanco, Alfonso; Borsellino, Giovanna; Boulais, Philip E.; Brinkman, Ryan R.; Bã¼scher, Martin; Busch, Dirk H.; Bushnell, Timothy P.; Cao, Xuetao; Cavani, Andrea; Chattopadhyay, Pratip K.; Cheng, Qingyu; Chow, Sue; Clerici, Mario; Cooke, Anne; Cosma, Antonio; Cosmi, Lorenzo; Cumano, Ana; Dang, Van Duc; Davies, Derek; De Biasi, Sara; Del Zotto, Genny; Della Bella, Silvia; Dellabona, Paolo; Deniz, Gã¼nnur; Dessing, Mark; Diefenbach, Andreas; Di Santo, James; Dieli, Francesco; Dolf, Andreas; Donnenberg, Vera S.; Dã¶rner, Thomas; Ehrhardt, Gã¶tz R. A.; Endl, Elmar; Engel, Pablo; Engelhardt, Britta; Esser, Charlotte; Everts, Bart; Falk, Christine S.; Fehniger, Todd A.; Filby, Andrew; Fillatreau, Simon; Follo, Marie; Fã¶rster, Irmgard; Foster, John; Foulds, Gemma A.; Frenette, Paul S.; Galbraith, David; Garbi, Natalio; Garcãa-godoy, Maria Dolores; Ghoreschi, Kamran; Gibellini, Lara; Goettlinger, Christoph; Goodyear, Carl S.; Gori, Andrea; Grogan, Jane; Gross, Mor; Grã¼tzkau, Andreas; Grummitt, Daryl; Hahn, Jonas; Hammer, Quirin; Hauser, Anja E.; Haviland, David L.; Hedley, David; Herrera, Guadalupe; Herrmann, Martin; Hiepe, Falk; Holland, Tristan; Hombrink, Pleun; Houston, Jessica P.; Hoyer, Bimba F.; Huang, Bo; Hunter, Christopher A.; Iannone, Anna; Jã¤ck, Hans-martin; Jã¡vega, Beatriz; Jonjic, Stipan; Juelke, Kerstin; Jung, Steffen; Kaiser, Toralf; Kalina, Tomas; Keller, Baerbel; Khan, Srijit; Kienhã¶fer, Deborah; Kroneis, Thomas; Kunkel, Dã©sirã©e; Kurts, Christian; Kvistborg, Pia; Lannigan, Joanne; Lantz, Olivier; Larbi, Anis; Leibundgut-landmann, Salome; Leipold, Michael D.; Levings, Megan K.; Litwin, Virginia; Liu, Yanling; Lohoff, Michael; Lombardi, Giovanna; Lopez, Lilly; Lovett-racke, Amy; Lubberts, Erik; Ludewig, Burkhard; Lugli, Enrico; Maecker, Holden T.; Martrus, Glã²ria; Matarese, Giuseppe; Mauerã¶der, Christian; Mcgrath, Mairi; Mcinnes, Iain; Mei, Henrik E.; Melchers, Fritz; Melzer, Susanne; Mielenz, Dirk; Mills, Kingston; Mjã¶sberg, Jenny; Moore, Jonni; Moran, Barry; Moretta, Alessandro; Moretta, Lorenzo; Mosmann, Tim R.; Mã¼ller, Susann; Mã¼ller, Werner; Mã¼nz, Christian; Multhoff, Gabriele; Munoz, Luis Enrique; Murphy, Kenneth M.; Nakayama, Toshinori; Nasi, Milena; Neudã¶rfl, Christine; Nolan, John; Nourshargh, Sussan; O'connor, Josã©-enrique; Ouyang, Wenjun; Oxenius, Annette; Palankar, Raghav; Panse, Isabel; Peterson, Pã¤rt; Peth, Christian; Petriz, Jordi; Philips, Daisy; Pickl, Winfried; Piconese, Silvia; Pinti, Marcello; Pockley, A. Graham; Podolska, Malgorzata Justyna; Pucillo, Carlo; Quataert, Sally A.; Radstake, Timothy R. D. J.; Rajwa, Bartek; Rebhahn, Jonathan A.; Recktenwald, Diether; Remmerswaal, Ester B. M.; Rezvani, Katy; Rico, Laura G.; Robinson, J. Paul; Romagnani, Chiara; Rubartelli, Anna; Ruland, Jã¼rgen; Sakaguchi, Shimon; Sala-de-oyanguren, Francisco; Samstag, Yvonne; Sanderson, Sharon; Sawitzki, Birgit; Scheffold, Alexander; Schiemann, Matthias; Schildberg, Frank; Schimisky, Esther; Schmid, Stephan A; Schmitt, Steffen; Schober, Kilian; Schã¼ler, Thomas; Schulz, Axel Ronald; Schumacher, Ton; Scotta, Cristiano; Shankey, T. Vincent; Shemer, Anat; Simon, Anna-katharina; Spidlen, Josef; Stall, Alan M.; Stark, Regina; Stehle, Christina; Stein, Merle; Steinmetz, Tobit; Stockinger, Hannes; Takahama, Yousuke; Tarnok, Attila; Tian, Zhigang; Toldi, Gergely; Tornack, Julia; Traggiai, Elisabetta; Trotter, Joe; Ulrich, Henning; Van Der Braber, Marlous; Van Lier, Renã© A. W.; Veldhoen, Marcello; Vento-asturias, Salvador; Vieira, Paulo; Voehringer, David; Volk, Hans-dieter; Von Volkmann, Konrad; Waisman, Ari; Walker, Rachael; Ward, Michael D.; Warnatz, Klaus; Warth, Sarah; Watson, James V.; Watzl, Carsten; Wegener, Leonie; Wi
abstract
Nessun abstract disponibile
2017
- HIV-DNA content in different CD4+ T-cell subsets correlates with CD4+ cell : CD8+ cell ratio or length of efficient treatment
[Articolo su rivista]
Gibellini, Lara; Pecorini, Simone; DE BIASI, Sara; Bianchini, Elena; Digaetano, Margherita; Pinti, Marcello; Carnevale, Gianluca; Borghi, Vanni; Guaraldi, Giovanni; Mussini, Cristina; Cossarizza, Andrea; Nasi, Milena
abstract
Objectives: HIV establishes a latent infection at different degrees within naïve (TN) or central (TCM) and effector memory (TEM) CD4+ T cell. Studying patients in whom HIV production was suppressed by combined antiretroviral therapy, our main aim was to find which factors are related or can influence intracellular viral reservoir in different CD4+ T-cell subsets. Methods: We enrolled 32 HIV+ patients successfully treated for more than 2 years, with a CD4+ T-cell count more than 500 cells/μl and plasma viremia undetectable from at least 1 year. Proviral HIV-DNA, the amount of cells expressing signal-joint T-cell receptor rearrangement excision circles and telomere length were quantified by droplet digital PCR in highly purified, sorted CD4+ T-cell subsets; plasma IL-7 and IL-15 were measured by ELISA. Results: HIV-DNA was significantly lower in TN cells compared with TCM or to TEM. Conversely, TN cells contained more signal-joint T-cell receptor rearrangement excision circles compared with TCM or to TEM; no appreciable changes were observed in telomere length. HIV-DNA content was significantly higher in TN and TCM cells, but not in TEM, from patients with shorter time of treatment, or in those with lower CD4+ : CD8+ ratio. Conclusion: Length of treatment or recovery of CD4+ : CD8+ ratio significantly influences viral reservoir in both TN and TCM. Measuring HIV-DNA in purified lymphocyte populations allows a better monitoring of HIV reservoir and could be useful for designing future eradication strategies.
2016
- . Pc-astakine mRNA levels decrease after both LPS injection and hemolymph withdrawals in the gastropod pest Pomacea canaliculata.
[Abstract in Rivista]
Accorsi, Alice; Gotting, Karen; Ross, Eric; Nasi, Milena; Ottaviani, Enzo; Sánchez Alvarado, Alejandro; Malagoli, Davide
abstract
The freshwater gastropod Pomacea canaliculata has recently been included among emerging pests in the EU. Hence, its immune system may be a promising target for pest control strategies. By analyzing a set of P. canaliculata transcriptomes, we have discovered an Astakine-1-like molecule (Pc-Astakine) in this organism. Astakine was first isolated as a hematopoietic cytokine in the crayfish Pacifastacus leniusculus (Pl-Astakine-1), and later found in arthropods and the bivalve Crassostrea gigas. Bioinformatic analyses show that Pc-Astakine is a 121 aa protein with a conserved distribution pattern of cysteines retrievable in both Pl-Astakine-1 and the vertebrate Prokineticin protein family. Pc-astakine is expressed at low levels in circulating hemocytes, but markedly in hemocyte reservoirs (e.g., ampulla) and hematopoietic tissue (pericardial fluid) suggesting a conserved role in hematopoiesis. We tested this potential role of Pc-Astakine in two ways. First, we measured RNA levels of this gene in immune-related tissues 24 h after injection of 50 μg of Escherichia coli-derived lipopolysaccharide (055:B5) (LPS). We observed a sharp decrease of Pc-astakine mRNA in all the analyzed organs, with Pc-astakine mRNA levels almost undetectable in anterior kidney (a hemocyte reservoir) when compared to sham-injected controls. Second, we defined the profile of Pc-astakine expression after a series of 4 hemolymph withdrawals performed within 72 h. As with the LPS-treatment, a general decrease of mRNA levels was observed, with the largest decrease observed in hematopoietic tissue and circulating hemocytes (reduced to 20% of the control). Altogether, we hypothesize that the decrease of Pc-astakine mRNA after both LPS injection and hemolymph withdrawal may be due to an increased rate of translation, rather than a reduction of gene expression. This is in part supported by similar observations in the oyster C. gigas in which massive translation of Cg-astakine is observed as a consequence of environment stress, including immune stimuli, and in P. leniusculus in which increased levels of Astakine in the plasma of LPS-injected animals have been related to an increased translational rate.
To our knowledge, this is the first report of an astakine-like molecule in gastropods and our data indicate a potential involvement of Pc-Astakine in both the immune response and hematopoiesis of the molluscan pest P. canaliculata.
2016
- Anti-TNF-α drugs differently affect the TNFa-sTNFR system and monocyte subsets in patients with psoriasis
[Articolo su rivista]
Gibellini, Lara; De Biasi, Sara; Bianchini, Elena; Bartolomeo, Regina; Fabiano, Antonella; Manfredini, Marco; Ferrar, Federica; Albertini, Giuseppe; Trenti, Tommaso; Nasi, Milena; Pinti, Marcello; Iannone, Anna; Salvarani, Carlo; Cossarizza, Andrea; Pellacani, Giovanni
abstract
TNF-a has a central role in the development and maintenance of psoriatic plaques, and its serum levels correlate with disease activity. Anti-TNF-a drugs are, however, ineffective in a relevant percentage of patients for reasons that are currently unknown. To understand whether the response to anti-TNF-a drugs is influenced by the production of anti-drug antibodies or by the modulation of the TNFa-TNFa receptor system, and to identify changes in monocyte phenotype and activity, we analysed 119 psoriatic patients who either responded or did not respond to different anti-TNF-a therapies (adalimumab, etanercept or infliximab), and measured plasma levels of TNF-a, TNF-a soluble receptors, drug and anti-drug antibodies. Moreover, we analyzed the production of TNF-a and TNF-α soluble receptors by peripheral blood mononuclear cells (PBMCs), and characterized different monocyte populations. We found that: i) the drug levels varied between responders and non-responders; ii) anti-infliximab antibodies were present in 15% of infliximab-treated patients, while anti-etanercept or anti-adalimumab antibodies were never detected; iii) plasma TNF-a levels were higher in patients treated with etanercept compared to patients treated with adalimumab or infliximab; iv) PBMCs from patients responding to adalimumab and etanercept produced more TNF-a and sTNFRII in vitro than patients responding to infliximab; v) PBMCs from patients not responding to infliximab produce higher levels of TNF-a and sTNFRII than patients responding to infliximab; vi) anti- TNF-a drugs significantly altered monocyte subsets. A complex remodelling of the TNFa-TNFa receptor system thus takes place in patients treated with anti-TNF-α drugs, that involves either the production of anti-drug antibodies or the modulation of monocyte phenotype or inflammatory activity.
2016
- Decreased circulating mtDNA levels in professional male volleyball players
[Articolo su rivista]
Nasi, Milena; Cristani, Alessandro; Pinti, Marcello; Lamberti, Igor; Gibellini, Lara; DE BIASI, Sara; Guazzaloca, Alessandro; Trenti, Tommaso; Cossarizza, Andrea
abstract
Purpose: Exercise exerts various effects on the immune system, and evidence is emerging on its anti-inflammatory effects; the mechanisms on the basis of these modifications are poorly understood. Mitochondrial DNA (mtDNA) released from damaged cells acts as a molecule containing the so-called damage-associated molecular patterns and can trigger sterile inflammation. Indeed, high plasma levels of mtDNA are associated to several inflammatory conditions and physiological aging and longevity. The authors evaluated plasma mtDNA in professional male volleyball players during seasonal training and the possible correlation between mtDNA levels and clinical parameters, body composition, and physical performance. Methods: Plasma mtDNA was quantified by real-time PCR every 2 mo in 12 professional volleyball players (PVPs) during 2 consecutive seasons. As comparison, 20 healthy nonathlete male volunteers (NAs) were analyzed. Results: The authors found lower levels of mtDNA in plasma of PVPs than in NAs. However, PVPs showed a decrease of circulating mtDNA only in the first season, while no appreciable variations were observed during the second season. No correlation was observed among mtDNA, hematochemical, and anthropometric parameters. Conclusions: Regular physical activity appeared associated with lower levels of circulating mtDNA, further confirming the protective, anti-inflammatory effect of exercise.
2016
- Effects of LPS injection on Pc-astakine
expression in the gastropod Pomacea
canaliculata
[Abstract in Rivista]
Benatti, Stefania; Accorsi, Alice; Nasi, Milena; Ottaviani, Enzo; Malagoli, Davide Malagoli
abstract
Astakine-1 is a prokineticin-containing factor
and the first hematopoietic cytokine described in
invertebrates. Astakine-1 was firstly retrieved in the
freshwater crayfish Pacifastacus leniusculus, and
recent experiments have demonstrated the
presence of astakine-like molecules also in insects
and molluscs, including the freshwater snail,
Pomacea canaliculata. In control conditions Pcastakine
is expressed in several organs, especially
in the ampulla (reservoir of hemocytes and potential
district of hemocyte maturation) and in the
pericardial fluid (i.e. the hematopoietic tissue). By
mean of qPCR experiments, we have analyzed the
effects of the injection of 50 µg LPS on the
expression of the gene Pc-astakine. Our
observations indicate that 24 h after the injection,
the major modification of the Pc-astakine expression
was evident in the anterior kidney, a potential
hemocyte reservoir, in which the expression of the
gene decreased to almost undetectable level. In the
pericardial fluid, ampulla and circulating hemocytes,
the expression of Pc-astakine dropped to less than
50 % with respect to the sham-injected control
snails. The drop in the amount of mRNA detected
by qPCR could reflect an increased rate of
translation and consequent degradation of the
available mRNA, rather than a decrease of the
transcription rate. Similarly, in the bivalve
Crassostrea gigas, it has been suggested that
accumulated Cg-astakine transcripts are largely
translated under some environment stress, including
immune stimuli. On the whole, our results indicate
that the expression of Pc-astakine and the
translation rate of its mRNA may be influenced by
immune stimuli, and support the hypothesis that PcAstakine
may be involved in Pomacea
hematopoiesis and/or may have immune-related
functions, as well.
2016
- Emerging role of Lon protease as a master regulator of mitochondrial functions
[Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Nasi, Milena; De Biasi, Sara; Bortolotti, Carlo Augusto; Iannone, Anna; Cossarizza, Andrea
abstract
Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
2016
- iNKT cells in secondary progressive multiple sclerosis patients display pro-inflammatory profiles
[Articolo su rivista]
DE BIASI, Sara; Simone, ANNA MARIA; Nasi, Milena; Bianchini, Elena; Ferraro, Diana; Vitetta, Francesca; Gibellini, Lara; Pinti, Marcello; DEL GIOVANE, Cinzia; Sola, Patrizia; Cossarizza, Andrea
abstract
Background. Multiple Sclerosis (MS), an autoimmune disease with neurodegeneration and inflammation, is characterized by several
alterations of different T cell subsets. However, few data exist on the role of iNKT lymphocytes.
Objective. To identify possible changes in the phenotype of iNKT cells in patients with different clinical forms of MS, and find alterations in their polyfunctionality (i.e., ability to produce simultaneously up to 4 cytokines such as IL‐17, TNF‐α, IFN‐γ, IL‐4). Methods. We studied a total of 165 patients, 91 with a Relapsing Remitting form [RR; 31 were treated with interferon (IFN)1‐β, 25 with natalizumab (Nat), 29 with glatiramer acetate (Gla); 17 were newly-diagnosed RR without treatment, 19 not active RR without treatment]. Forty-four patients had a Progressive MS: 20 Primary Progressive (PP), 24 Secondary Progressive (SP). A total of 55 age- and sex-matched subjects represented healthy controls (CTR). Among fresh peripheral blood mononuclear cells (PBMC) iNKT cells were identified by flow cytometry. Moreover, the capability of iNKT cells to produce different cytokines (IL‐17, TNF‐α, IFN‐γ, and IL‐4) after in vitro stimulation were evaluated in 18 RR (11 treated with Nat and 7 with IFN), 4 PP, 6 SP and 16 CTR. Results. No main differences were found in iNKT cell phenotype among MS patients with different MS forms, or during different treatments. However, the polyfunctional response of iNKT cells showed Th1 and Th17 profiles. This was well evident in patients with secondary progressive form, who are characterized by high levels of inflammation and neurodegeneration, and exhibited a sustained increase in the production of Th17 cytokines. Patients treated with natalizumab displayed lower levels of iNKT cells producing IL‐17, TNF‐α and IFN‐γ.
Conclusion. Our data suggest that the progressive phase of the disease is characterized by permanent iNKT activation and a skewing towards an inflammatory phenotype. Compared to other treatments, natalizumab was able to modulate iNKT cell function.
2016
- Mitochondrial proteases as emerging pharmacological targets
[Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello
abstract
The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions.
2016
- Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry
[Articolo su rivista]
DE BIASI, Sara; Gibellini, Lara; Bianchini, Elena; Nasi, Milena; Pinti, Marcello; Salvioli, Stefano; Cossarizza, Andrea
abstract
Reactive oxygen species (ROS) are constantly produced in cells, mainly by mitochondria, as a consequence of aerobic respiration. Most ROS derive from superoxide, which is rapidly converted to hydrogen peroxide. ROS are involved in the regulation of several physiological and pathological processes, and the possibility to measure them simultaneously is needed, when the redox status of the cells is modified by experimental/biological conditions. Flow cytometry is the main technology that generates multiple information at the single cell level in a high-throughput manner, and gives rapid and quantitative measurements of different ROS with high sensitivity and reproducibility. Here, we describe a novel approach to detect simultaneously mitochondrial hydrogen peroxide and mitochondrial superoxide in living cells. The staining has been performed by using the fluorescent dyes MitoSOX Red Mitochondrial Superoxide Indicator, Mitochondria Peroxy Yellow 1, Annexin-V Pacific Blue conjugate, TO-PRO-3 iodide, anti-CD4-APC-Cy7 and -CD8-Pacific Orange mAbs. We used this approach to quantify mitochondrial ROS in CD4+ and CD8+ T cells form patients affected by Down syndrome and age- and sex-matched healthy donors.
2016
- Th1 and Th17 pro-inflammatory profile characterizes iNKT cells in virologically suppressed HIV+ patients with low CD4/CD8 ratio
[Articolo su rivista]
DE BIASI, Sara; Bianchini, Elena; Nasi, Milena; Digaetano, Margherita; Gibellini, Lara; Carnevale, Gianluca; Borghi, Vanni; Guaraldi, Giovanni; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract
INTRODUCTION:: Scanty data exist on the phenotype and functionality of invariant natural killer T (iNKT) cells in HIV+ patients (pts). METHODS:: By flow cytometry, we studied iNKT cells from 54 HIV+ pts who started combined antiretroviral therapy (cART) and had undetectable viral load for >1 year. Twenty-five maintained a CD4/CD8 ratio <0.4, while 29 reached a ratio >1.1; 32 age- and sex-matched subjects were healthy controls (CTR). RESULTS:: Pts with low ratio had lower percentage of CD4+ iNKT cells compared to pts with high ratio, and higher CD8+ iNKT cell percentage; double negative (DN) iNKT cells were lower in HIV+ pts compared to CTR. Pts with low ratio had higher percentage of CD4+ and DN iNKT cells expressing CD38 and HLA-DR compared to pts with high ratio. CD4+ iNKT cells expressing PD-1 were higher in pts with CD4/CD8 ratio <0.4, while DN iNKT cells expressing PD-1 were lower compared to pts with ratio >1.1. Pts with low ratio had higher CD4+ iNKT cells producing IL-17, CD8+ iNKT cells producing IFN-γ, TNF-α or IFN-γ plus TNF-α, and DN iNKT cells producing IL-17 or IL-17 plus IFN-γ compared to CTR. Activated CD4+ (or CD8+) T cells correlated with activated CD4+ (or CD8+) iNKT cells, as well as the percentages of CD4+ (or CD8+) T cells expressing PD-1 was correlated to that of CD4+ (or CD8+) iNKT cells expressing PD-1. CONCLUSIONS:: Low CD4/CD8 ratio despite effective cART is associated with altered iNKT cell subsets, enhanced activation and prominent Th1/Th17 pro-inflammatory profile.
2015
- Analysis of inflammasomes and antiviral sensing components reveals decreased expression of NLRX1 in HIV-positive patients assuming efficient antiretroviral therapy
[Articolo su rivista]
Nasi, Milena; DE BIASI, Sara; Bianchini, Elena; Digaetano, Margherita; Pinti, Marcello; Gibellini, Lara; Pecorini, Simone; Carnevale, Gianluca; Guaraldi, Giovanni; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea
abstract
Objective: Few studies have investigated the importance of different components of the inflammasome system and of innate mitochondrial sensing (IMS) pathways in HIV infection and its treatment. We analysed the expression of several components of the inflammasome and of the IMS in HIV-positive patients taking successful combination antiretroviral therapy (cART). Methods: We enrolled 20 HIV-positive patients under cART, who achieved viral suppression since at least 10 months and 20 age and sex-matched healthy donors. By RT-PCR, using peripheral blood mononuclear cells (PBMCs), we quantified the mRNA expression of 16 genes involved in inflammasome activation and regulation (AIM2, NAIP, PYCARD, CASP1, CASP5, NLRP6, NLRP1, NLRP3, TXNIP, BCL2, NLRC4, PANX1, P2RX7, IL-18, IL-1β, SUGT1) and eight genes involved in IMS (MFN2, MFN1, cGAS, RIG-I, MAVS, NLRX1, RAB32, STING). Results: Compared with controls, HIV-positive patients showed significantly lower mRNA levels of the mitochondrial protein NLRX1, which plays a key role in regulating apoptotic cell death; main PBMC subpopulations behave in a similar manner. No differences were observed in the expression of inflammasome components, which however showed complex correlations. Conclusion: The decreased level of NLRX1 in HIV infection could suggest that the virus is able to downregulate mechanisms linked to triggering of cell death in several immune cell types. The fact that HIV-positive patients did not show altered expression of inflammasome components, nor of most genes involved in IMS, suggests that the infection and/or the chronic immune activation does not influence the transcriptional machinery of innate mechanisms able to trigger inflammation at different levels.
2015
- Different origin of adipogenic stem cells influences the response to antiretroviral drugs
[Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Carnevale, Gianluca; Pisciotta, Alessandra; Bianchini, Elena; Bartolomeo, Regina; Polo, Miriam; DE POL, Anto; Pinti, Marcello; Cossarizza, Andrea
abstract
Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50μM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin.
2015
- Inhibition of Lon protease by triterpenoids alters mitochondria and is associated to cell death in human cancer cells
[Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Bartolomeo, Regina; De Biasi, Sara; Cormio, Antonella; Musicco, Clara; Carnevale, Gianluca; Pecorini, Simone; Nasi, Milena; De Pol, Anto; Cossarizza, Andrea
abstract
Mitochondrial Lon protease (Lon) regulates several mitochondrial functions, and is inhibited by the anticancer molecule triterpenoid 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), or by its C-28 methyl ester derivative (CDDO-Me). To analyze the mechanism of action of triterpenoids, we investigated intramitochondrial reactive oxygen species (ROS), mitochondrial membrane potential, mitochondrial mass, mitochondrial dynamics and morphology, and Lon proteolytic activity in RKO human colon cancer cells, in HepG2 hepatocarcinoma cells and in MCF7 breast carcinoma cells. We found that CDDO and CDDO-Me are potent stressors for mitochondria in cancer cells, rather than normal non-transformed cells. In particular, they: i) cause depolarization; ii) increase mitochondrial ROS, iii) alter mitochondrial morphology and proteins involved in mitochondrial dynamics; iv) affect the levels of Lon and those of aconitase and human transcription factor A, which are targets of Lon activity; v) increase level of protein carbonyls in mitochondria; vi) lead to intrinsic apoptosis. The overexpression of Lon can rescue cells from cell death, providing an additional evidence on the role of Lon in conditions of excessive stress load.
2015
- Natural Compounds Modulating Mitochondrial Functions
[Articolo su rivista]
Gibellini, Lara; Bianchini, Elena; DE BIASI, Sara; Nasi, Milena; Cossarizza, Andrea; Pinti, Marcello
abstract
Mitochondria are organelles responsible for several crucial cell functions, including respiration, oxidative phosphorylation, and regulation of apoptosis; they are also the main intracellular source of reactive oxygen species (ROS). In the last years, a particular interest has been devoted to studying the effects on mitochondria of natural compounds of vegetal origin, quercetin (Qu), resveratrol (RSV), and curcumin (Cur) being the most studied molecules. All these natural compounds modulate mitochondrial functions by inhibiting organelle enzymes or metabolic pathways (such as oxidative phosphorylation), by altering the production of mitochondrial ROS and by modulating the activity of transcription factors which regulate the expression of mitochondrial proteins. While Qu displays both pro- and antioxidant activities, RSV and Cur are strong antioxidant, as they efficiently scavenge mitochondrial ROS and upregulate antioxidant transcriptional programmes in cells. All the three compounds display a proapoptotic activity, mediated by the capability to directly cause the release of cytochrome c from mitochondria or indirectly by upregulating the expression of proapoptotic proteins of Bcl-2 family and downregulating antiapoptotic proteins. Interestingly, these effects are particularly evident on proliferating cancer cells and can have important therapeutic implications.
2015
- Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology
[Articolo su rivista]
Nasi, Milena; De Biasi, Sara; Bianchini, Elena; Gibellini, Lara; Pinti, Marcello; Scacchetti, Tiziana; Trenti, Tommaso; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea
abstract
An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the "gold standard" for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the "CD4% Count Kit-Dry".
2014
- Aging with HIV infection: a journey to the center of inflammAIDS, immunosenescence and neuroHIV
[Articolo su rivista]
Nasi, Milena; Pinti, Marcello; DE BIASI, Sara; Gibellini, Lara; Ferraro, Diana; Mussini, Cristina; Cossarizza, Andrea
abstract
In the last years, a significant improvement in life expectancy of HIV+ patients has been observed in Western countries. The parallel increase in the mean age of these patients causes a parallel increase in the frequency of non-AIDS related complications (i.e., neurocognitive, cardiovascular, liver and kidney diseases, metabolic syndrome, osteoporosis, non-HIV associated cancers, among others), even when antiviral treatment is successful. Immune activation and persistent inflammation characterizes both HIV infection and physiological aging, and both conditions share common detrimental pathways that lead to early immunosenescence. Furthermore, HIV-associated neurocognitive disorders represent important consequences of the infection. The persistent systemic immune activation, the continuous migration of activated monocytes to the central nervous system and progressive patients' aging contribute to develop neuronal injuries, that are in turn linked to HIV-associated neurocognitive disorders, which can persist despite successful antiretroviral treatment.
2014
- Circulating mitochondrial DNA increases with age and is a familiar trait: implications for "inflamm-aging"
[Articolo su rivista]
Pinti, Marcello; Cevenini, E; Nasi, Milena; De Biasi, Sara; Salvioli, S; Monti, Daniela; Benatti, Stefania; Gibellini, Lara; Cotichini, R; Stazi, Ma; Trenti, T; Franceschi, C; Cossarizza, Andrea
abstract
Mitochondrial components, including mitochondrial DNA (mtDNA), when released extracellularly, can act as "damage-associated molecular pattern" (DAMP) agents and cause inflammation. As many elderly people are characterized by a low-grade, chronic inflammatory status defined "inflamm-aging", we evaluated if circulating mtDNA can contribute to this phenomenon. Eight hundred and thirty-one Caucasian subjects were enrolled in the study, including 429 siblings aged 90-104 years (90+ siblings). MtDNA plasma levels increased gradually after the fifth decade of life. In 90+ subjects, mtDNA values of two members of the same sibling relationship were directly correlated, suggesting a role for familiar/genetic background in controlling the levels of circulating mtDNA. The subjects with the highest mtDNA plasma levels had the highest amounts of TNF-??, IL-6, RANTES and IL-1ra; the subjects with the lowest mtDNA levels had the lowest levels of the same cytokines. In vitro stimulation of monocytes with mtDNA concentrations similar to the highest levels observed in vivo resulted in an increased production of TNF-??, suggesting that mtDNA can modulate the production of proinflammatory cytokines. Our findings therefore show that circulating mtDNA increases with age, and can significantly contribute to the maintenance of the low grade, chronic inflammation observed in elderly people This article is protected by copyright. All rights reserved.
2014
- Persistent inflammation in HIV infection: Established concepts, new perspectives.
[Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Mussini, Cristina; Cossarizza, Andrea
abstract
Immune activation is now considered a main driving force for the progressive immune failure in HIV infection. During the early phases of infection, a rapid depletion of gastrointestinal CD4+ T cells occurs that is followed by a deterioration of the gut epithelium and by the subsequent translocation of microbial products into the blood. Activation of innate immunity results in massive production of proinflammatory cytokines, which can trigger activation induced cell death phenomena among T lymphocytes. Moreover, persistent antigenic stimulation and inflammatory status causes immune exhaustion. The chronic immune activation also damages lymphoid tissue architecture, so contributing to the impairment of immune reconstitution. Recently, new mechanisms were identified, so opening new perspective on the innate immune sensing in HIV-1 infection. Cell death is followed by the release of molecules containing "damage-associated molecular patterns", that trigger a potent innate immune response through the engagement of Toll-like receptors. Then, also different types of HIV-related nucleic acids can act as potent stimulators of innate immunity. All these events contribute to the loss of T cell homeostatic regulation and to the failure of adaptive immunity.
2014
- Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.
[Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Boraldi, Federica; Giorgio, Valentina; Bernardi, Paolo; Bartolomeo, Regina; Nasi, Milena; DE BIASI, Sara; Missiroli, Sonia; Carnevale, Gianluca; Losi, Lorena; Tesei, Anna; Pinton, Paolo; Quaglino, Daniela; Cossarizza, Andrea
abstract
Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.-Gibellini, L., Pinti, M., Boraldi, F., Giorgio, V., Bernardi, P., Bartolomeo, R., Nasi, M., De Biasi, S., Missiroli, S., Carnevale, G., Losi, L., Tesei, A., Pinton, P., Quaglino, D., Cossarizza, A. Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.
2014
- Sirtuin 3 interacts with Lon protease and regulates its acetylation status.
[Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Beretti, Francesca; Pierri, Cl; Onofrio, A; Riccio, Massimo; Carnevale, Gianluca; DE BIASI, Sara; Nasi, Milena; Torelli, F; Boraldi, Federica; DE POL, Anto; Cossarizza, Andrea
abstract
Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.
2014
- Successful treatment of HIV-1 infection increases the expression of a novel, short transcript for IL-18 receptor α chain
[Articolo su rivista]
Nasi, Milena; Alboni, Silvia; Pinti, Marcello; Tascedda, Fabio; Benatti, Cristina; Benatti, Stefania; Gibellini, Lara; DE BIASI, Sara; Borghi, Vanni; Brunello, Nicoletta; Mussini, Cristina; Cossarizza, Andrea
abstract
The importance of interleukin (IL)-18 in mediating immune activation during HIV infection has recently emerged. IL-18 activity is regulated by its receptor (IL-18R), formed by an α and a β chain, the IL-18-binding protein, and the newly identified shorter isoforms of both IL-18R chains. We evaluated gene expression of the IL-18/IL-18R system in peripheral blood mononuclear cells from HIV+ patients. Compared with healthy donors, IL-18 expression decreased in patients with primary infection. The IL-18Rα short transcript expression was strongly upregulated by successful highly active antiretroviral therapy. HIV progression and its treatment can influence the expression of different components of the complex IL-18/IL-18R system.
2013
- Cytometry, immunology, and HIV infection: Three decades of strong interactions.
[Articolo su rivista]
Cossarizza, Andrea; DE BIASI, Sara; Gibellini, Lara; Bianchini, E; Bartolomeo, Regina; Nasi, Milena; Mussini, Cristina; Pinti, Marcello
abstract
Flow cytometry (FCM) has been extensively used to investigate immunological changes that occur from infection with the human immunodeficiency virus (HIV). This review describes some of the most relevant cellular and molecular changes in the immune system that can be detected by FCM during HIV infection. Finally, it will be discussed how this technology has facilitated the understanding not only of the biology of the virus but also of the mechanisms that the immune system activates to fight HIV and is allowing to monitor the efficacy of antiretroviral therapy.
2013
- Novel genetic association of TNF-α-238 and PDCD1-7209 polymorphisms with long-term non-progressive HIV-1 infection.
[Articolo su rivista]
Nasi, Milena; Riva, A; Borghi, V; D'Amico, Roberto; DEL GIOVANE, Cinzia; Casoli, C; Galli, M; Vicenzi, E; Gibellini, Lara; DE BIASI, Sara; Clerici, M; Mussini, Cristina; Cossarizza, Andrea; Pinti, Marcello
abstract
About 2-5% of HIV-1-infected subjects, defined as long-term non-progressors (LTNPs), remain immunologically stable for a long time without treatment. The factors governing this condition are known only in part, and include genetic factors. Thus, we studied 20 polymorphisms of 15 genes encoding proinflammatory and immunoregulatory cytokines, chemokines and their receptors, genes involved in apoptosis, and the gene HCP5. METHODS: We analyzed 47 Caucasian LTNPs infected for >9 years, compared with 131 HIV-1-infected Caucasian patients defined as 'usual progressors'. The genotypes were determined by methods based upon PCR, and the statistical analysis was performed by univariate logistic regression. RESULTS: The well-known CCR5Δ32 del32 allele, the cell death-related TNF-α-238 A and PDCD1-7209 T alleles, and HCP5 rs2395029 G, a non-coding protein associated with the HLA-B*5701, were found positively associated with the LTNP condition. No association was observed for other single nucleotide polymorphisms (SDF-1-801, IL-10-592, MCP-1-2518, CX3CR1 V249I, CCR2V64I, RANTES-403, IL-2-330, IL-1β-511, IL-4-590, FASL IVS3nt-169, FAS-670, FAS-1377, FASL IVS2nt-124, PDCD1-7146, MMP-7-181, and MMP7-153). CONCLUSIONS: The novel genetic associations between allelic variants of genes TNF-α-238 and PDCD1-7209 with the LTNP condition underline the importance of host genetic factors in the progression of HIV-1 infection and in immunological preservation.
2012
- Immunological advantages of everolimus versus cyclosporin A in liver-transplanted recipients, as revealed by polychromatic flow cytometry.
[Articolo su rivista]
Roat, Erika; DE BIASI, Sara; Bertoncelli, Linda; Rompianesi, Gianluca; Nasi, Milena; Gibellini, Lara; Pinti, Marcello; DEL GIOVANE, Cinzia; A., Zanella; DI BENEDETTO, Fabrizio; Gerunda, Giorgio Enrico; Cossarizza, Andrea
abstract
Several immunosuppressive drugs with different mechanisms of action are available to inhibit organ rejection after transplant. We analyzed different phenotypic and functional immunological parameters in liver-transplanted patients who received cyclosporin A (CsA) or Everolimus (Evr). In peripheral blood mononuclear cells (PBMC) from 29 subjects receiving a liver transplant and treated with two different immunosuppressive regimens, we analyzed T cell activation and differentiation, regulatory T cells (Tregs) and Tregs expressing homing receptors such as the chemokine receptor CXCR3. T cell polyfunctionality was studied by stimulating cells with the superantigen staphylococcal enterotoxin B (SEB), and measuring the simultaneous production of interleukin (IL)-2 and interferon (IFN)-γ, along with the expression of a marker of cytotoxicity such as CD107a. The analyses were performed by polychromatic flow cytometry before transplantation, and at different time points, up to 220 days after transplant. Patients taking Evr had a higher percentage of total CD4⁺ and naïve CD4⁺ T cells than those treated with CsA; the percentage of CD8⁺ T cells was lower, but the frequency of naïve CD8⁺ T cells higher. Patients taking Evr showed a significantly higher percentage of Tregs, and Tregs expressing CXCR3. After stimulation with SEB, CD8⁺ T cells from Evr-treated patients displayed a lower total response, and less IFN-γ producing cells. The effects on the immune system, such as the preservation of the naïve T cell pool and the expansion of Tregs (that are extremely useful in inhibiting organ rejection), along with the higher tolerability of Evr, suggest that this drug can be safely used after liver transplantation, and likely offers immunological advantages.
2012
- Mitochondrial DNA haplogroups and incidence of lipodystrophy in HIV-infected patients on long-term antiretroviral therapy.
[Articolo su rivista]
De Luca, A; Nasi, Milena; Di Giambenedetto, S; Cozzi Lepri, A; Pinti, Marcello; Marzocchetti, A; Mussini, Cristina; Fabbiani, M; Bracciale, L; Cauda, R; Cossarizza, Andrea
abstract
We investigated the rates of lipodystrophy events, according to mitochondrial DNA haplogroup, in 187 patients starting combination antiretroviral therapy and following it. Incidence rates of lipoatrophy and fat accumulation were 8.2 and 4.8 per 100 person-years of follow-up, respectively. In multivariable models, patients with haplogroup K were at higher risk of any lipodystrophy [adjusted relative risk (aRR) 4.02, P = 0.0009], lipoatrophy (competing-risk aRR 2.42, P = 0.09; cause-specific aRR 2.99, P = 0.031), and fat accumulation (competing-risk aRR, 2.63, P = 0.11; cause-specific aRR 5.27, P = 0.019) than those with haplogroup H. Mitochondrial haplogroups may explain part of the genetic predisposition to lipodystrophy during combination antiretroviral therapy.
2012
- T cell activation but not polyfunctionality after primary HIV infection predicts control of viral load and length of the time without therapy.
[Articolo su rivista]
Cossarizza, Andrea; Bertoncelli, Linda; Nemes, Elisa; Lugli, Enrico; Pinti, Marcello; Nasi, Milena; DE BIASI, Sara; Gibellini, Lara; Montagna, Jp; Vecchia, M; Manzini, Lisa; Meschiari, Marianna; Borghi, Valentina; Guaraldi, Giovanni; Mussini, Cristina
abstract
Immune changes occurring after primary HIV infection (PHI) have a pivotal relevance. Our objective was to characterize the polyfunctionality of immune response triggered by PHI, and to characterize immune activation and regulatory T cells, correlating such features to disease progression.
PATIENTS AND METHODS:
We followed 11 patients experiencing PHI for 4 years. By polychromatic flow cytometry, we studied every month, for the first 6 months, T lymphocyte polyfunctionality after cell stimulation with peptides derived from HIV-1 gag and nef. Tregs were identified by flow cytometry, and T cell activation studied by CD38 and HLA-DR expression.
RESULTS:
An increase of anti-gag and anti-nef CD8+ specific T cells was observed 3 months after PHI; however, truly polyfunctional T cells, also able to produce IL-2, were never found. No gross changes in Tregs were present. T lymphocyte activation was maximal 1 and 2 months after PHI, and significantly decreased in the following period. The level of activation two months after PHI was strictly correlated to the plasma viral load 1 year after infection, and significantly influenced the length of period without therapy. Indeed, 80% of patients with less than the median value of activated CD8+ (15.5%) or CD4+ (0.9%) T cells remained free of therapy for >46 months, while all patients over the median value had to start treatment within 26 months.
CONCLUSIONS:
T cell activation after PHI, more than T cell polyfunctionality or Tregs, is a predictive marker for the control of viral load and for the time required to start treatment.
2012
- The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes
[Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Pinti, Marcello; Nasi, Milena; Riccio, Massimo; Carnevale, Gianluca; Cavallini, Gian Maria; F. J., Sala De Oyanguren; J. E., O’Connor; Mussini, Cristina; DE POL, Anto; Cossarizza, Andrea
abstract
Background: The association betweenHAARTand lipodystrophy iswell established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells. Methods: SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10–200mmol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2-) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2 - were studied by flow cytometry and multispectral imaging flow cytometry. Results: In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization. Conclusion: Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.
2011
- CD4+ T-cell differentiation, regulatory T cells and gag-specific T lymphocytes are unaffected by CD4-guided treatment interruption and therapy resumption.
[Articolo su rivista]
Nemes, Elisa; Lugli, Enrico; Bertoncelli, Linda; Nasi, Milena; Pinti, Marcello; Manzini, S; Prati, Francesca; Manzini, Lisa; DEL GIOVANE, Cinzia; D'Amico, Roberto; Cossarizza, Andrea; Mussini, Cristina
abstract
OBJECTIVES:Despite limiting exposure to antiretroviral drugs, structured treatment interruptions can influence multiple aspects of T-cell immunity, particularly those regarding CD4(+) T lymphocytes. We evaluated the impact of CD4-guided treatment interruption (CD4-GTI) and treatment resumption on regulatory T cells (Tregs), T-lymphocyte activation, differentiation and polyfunctional gag-specific response.METHODS:Patients were analyzed just prior to treatment interruption, at 2 and 6 months after treatment interruption, just prior to treatment resumption and at 2 and 6 months after treatment resumption. Thawed peripheral blood mononuclear cells were stained immediately for phenotype analysis or stimulated with HIV-gag peptides and analyzed by polychromatic flow cytometry.RESULTS:Treatment interruption resulted in a CD4(+) cell count decrease and plasma viral load (pVL) increase, but did not preclude a good immune reconstitution and a complete suppression of pVL after treatment resumption. Treatment interruption did not influence CD4(+) T-cell differentiation and Treg subsets. During treatment interruption, gag-specific CD4(+) T cells were not lost, although the frequency of HIV-specific CD8(+) cells increased. Most gag-specific CD4(+) T cells were potentially cytotoxic (CD107a(+)) and were not influenced by pVL or by HAART. Most helper (CD154(+)) gag-specific CD4(+) T lymphocytes did not produce interferon-γ or interleukin-2.CONCLUSION:CD4-GTI did not cause depletion of memory cells, Tregs or HIV-specific CD4(+) cells and, on the contrary, could induce HIV-specific responses. If guided by CD4(+) T-cell count, treatment interruption does not provoke irreversible immune damages.
2011
- Decreased mitochondrial DNA content in subcutaneous fat from HIV-infected women taking antiretroviral therapy as measured at delivery.
[Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Chiesa, E; Fiore, S; Manzini, S; DEL GIOVANE, Cinzia; D'Amico, Roberto; Palai, N; Campatelli, C; Sabbatini, F; Roccio, M; Tibaldi, C; Masuelli, G; Mussini, Cristina; Ferrazzi, E; d'Arminio Monforte, A; Cossarizza, Andrea
abstract
BACKGROUND: Increasing numbers of pregnant HIV-positive women are receiving combination antiretroviral regimens for preventing mother-to-child virus transmission or for treating the infection itself. Several studies have demonstrated that nucleoside reverse transcriptase inhibitors (NRTIs) induce mitochondrial toxicity by several mechanisms, including depletion of mitochondrial DNA (mtDNA). By the quantification of mtDNA levels, we studied mitochondrial toxicity in HIV-positive women at delivery and the possible correlations with antiretroviral regimens, viroimmunological and metabolic parameters.METHODS: We analysed 68 HIV-positive women enrolled in the Italian Prospective Cohort Study on Efficacy and Toxicity of Antiretroviral in Pregnancy (TARGET Study); all were taking ≥1 NRTI. We quantified mtDNA copies per cell in subcutaneous fat samples collected during delivery. At the 3rd, 6th and 9th month of pregnancy, we collected data concerning CD4(+) T-cell count, plasma HIV RNA, total and high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and triglycerides. As a control, we analysed mtDNA levels in abdominal subcutaneous fat samples from 23 HIV-seronegative women at delivery.RESULTS: mtDNA content was significantly lower in HIV-infected women when compared with HIV-negative controls. mtDNA content varied independently from viroimmunological, lipid and glucose parameters at the different months, with the exceptions of triglycerides at the 9th month and of HDL at the 6th month of pregnancy.CONCLUSIONS: In subcutaneous tissue from women taking NRTI-based antiretroviral regimens, we observed a significant decrease of mtDNA content, compared with uninfected women not on antiviral treatment. Moreover, a significant correlation was noted between mtDNA content and HDL cholesterol and triglycerides.
2011
- EXTRACELLULAR MITOCHONDRIAL DNA PLASMA LEVELS INCREASE DURING AGING AND LONGEVITY: IMPLICATIONS FOR "INFLAMMAGING"
[Abstract in Rivista]
Cossarizza, Andrea; Cevenini, E.; Stazi, M. A.; Cotichini, R.; Monti, Daniela; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Benatti, Stefania; Pinti, Marcello; Franceschi, Claudio
abstract
Mitochondrial (mt) DNA or degraded mitochondrial peptides are involved in the pathogenesis of the systemic inflammatory response syndrome (SIRS), a condition that often affects patients who survive a trauma and that resembles sepsis. Indeed, SIRS is due, at least partially, to molecules called "damage-associated molecular patterns" (DAMPs), a family of conserved molecules, conceptually similar to PAMPs, that includes hyaluronan fragments, heat shock proteins, S100 proteins, beta-amyloid, uric acid, IL-1 alpha, IL-33, and the DNA-binding nuclear protein HMGB1. DAMPs can trigger an innate immune response, causing inflammation through the engagement of several TLRs. Mitochondrial DAMPs (MTDs) can be released by damaged tissues, and are highly present in plasma of patients with SIRS. MTDs are represented by formyl peptides, that bind the formyl peptide receptor-1 (FPR-1), and mtDNA, that binds TLR-9, whose activation causes a potent inflammatory reaction.In the last years, we have shown that the production of proinflammatory cytokines is highly increased during the aging process, which is characterized by the accumulation of cellular and molecular defects, and involutive phenomena occurring concomitantly to a hyperstimulation of both innate and adaptive immunity (accumulation of expanded clones of memory and effector T cells, shrinkage of the TCR repertoire, progressive activation of macrophages). These phenomena result in a low-grade, chronic state of inflammation defined as “inflammaging”.To investigate the possible role of extracellular mtDNA during inflammaging, by an original real time PCR assay we have measured plasma levels of mtDNA in individuals who have been followed for 5 years: 429 siblings >90 years old (including 20 ultracentenarians) and 231 controls (50-75 y.o.). Moreover, we have studied 50 young donors (<30 years). A highly significant age-related increase of mtDNA plasma levels was observed. In donors >90 years, mtDNA plasma levels were significantly similar within families, suggesting the existence of a possible genetic component that controls this parameter. mtDNA was not correlated to classical inflammatory markers (CRP, VES), nor to main hematochemical conventional risk factors (among which ALT, albumin, cholesterol and glycemia). Finally, individuals who died within one year from the plasma collection had a lower level of mtDNA, while those who survived >5 years had much higher mtDNA plasma content.We can hypothesize that the age-related increase in plasma mtDNA could be either a marker of an optimal elimination of potentially dangerous cells, or the result of a complex remodeling of the entire organism, that makes use of potentially proinflammatory molecules like MTDs.
2011
- Functional characterization of the promoter of the human Lon protease gene.
[Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Roat, Erika; O'Connor, Je; Cossarizza, Andrea
abstract
Lon, a nuclear-encoded mitochondrial enzyme, degrades oxidized proteins of the mitochondrial (mt) matrix, and participates in the replication of mtDNA. Lon is upregulated in the presence of substances such as stavudine (d4T), D-deoxyribose (dRib), that increase the intracellular reactive oxygen species (ROS) levels, or in the presence of H(2)O(2.) Here we show the promoter region -623/+1 is essential for response to ROS, and that in SW872, HepG2 and WI-38 cell lines the region -1230/-623 represses transcription, while the region -2023/-1230 increases promoter activity. D4T upregulates Lon promoter activity in all cell lines while dRib upregulates Lon mainly in HepG2 cells, and in shorter incubation times. These data confirm that Lon can be considered a stress responsive protein.
2011
- HIV-1 Infection and the Aging of the Immune System: Facts, Similarities and Perspectives
[Articolo su rivista]
DE BIASI, Sara; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; Bertoncelli, Linda; Manzini, Serena; Mussini, Cristina; Cossarizza, Andrea
abstract
During infection with the human immunodeficiency type-1 virus (HIV), the immune system has to cope with the exposure to an unexpected number of different and new antigens that are generated by continuous mutations of the virus. This phenomenon causes a profound derangement of the immune response, which is similar to that defined immunosenescence, a complex remodeling, whereby clonotypical immunity deteriorates, and ancestral and innate immunity is largely preserved. Either in HIV+ patients or in elderly individuals, the lifelong chronic antigenic stress, along with the involution of the thymus, causes the accumulation of memory/effector T cells and the exhaustion of naïve T cells. Furthermore, in both these conditions a chronic inflammatory status exists in the aging process, which has been defined as "inflammaging" and is characterized by an enhanced production of proinflammatory cytokines. In this review, we will underline the similarities that exist between immunological changes present during the physiological aging process and HIV infection. © 2011.
2011
- INCREASED LEVELS OF PLASMATIC MITOCHONDRIAL DNA DURING HIV INFECTION REVEAL A NEW ROLE FOR MITOCHONDRIAL DAMAGE-ASSOCIATED MOLECULAR PATTERNS
[Abstract in Rivista]
Cossarizza, Andrea; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Montagna, J. P.; Bertoncelli, Linda; Bisi, Luca; Manzini, Lisa; Benatti, Stefania; Borghi, V.; Mussini, Cristina
abstract
Recently, it has been shown that mtDNA or degraded mitochondrial peptides can act as “damage-associated molecular patterns" (DAMPs), that are conceptually and functionally similar to PAMPs. Mitochondrial DAMPs, defined MTDs, can be involved in the pathogenesis of the systemic inflammatory response syndrome (SIRS), a condition that often affects patients who survive a trauma, characterized by the presence of shock and compromised function of several organs. During SIRS, mtDNA released from damaged or dead cells can bind TLR-9, whose activation causes a potent inflammatory reaction, with the production of proinflammatory cytokines. Since HIV infection is characterized by a proinflammatory status, and by a hyperproduction of proinflammatory cytokines, we asked whether soluble mtDNA circulating in the plasma could play a role in determining such condition.Thus, we have studied plasma levels of mtDNA in HIV+ patients showing a different course of the infection, and have correlated such levels to the activation of the immune system and to the plasma viremia. We analyzed individuals during acute, primary HIV infection (AHI), patients with an advanced infection (including those with full blown acquired immunodeficiency syndrome, AIDS) but still naive for antiretroviral therapy (ART), and those defined “long term non progressors” (LTNPs), who had been infected since at least 8 years, always out of treatment, but with a normal number of CD4+ T cells, a low grade of apoptosis and a good immunological control of the virus.In all HIV+ patients but LTNP plasma levels of mtDNA were significantly higher than in healthy controls. Furthermore, in naive patients, 6 months of efficient ART (able to increase CD4+ T cell count, decrease viral load and reduce T cell activation) did not modify mtDNA plasma levels. These levels were not correlated to CD4+ T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA not only as a molecule able to trigger inflammation, but also as a novel biomarker of virus-induced damage.The identification of the role of MTDs could relevant not only to identify possible new biomarkers of disease progression, but also in designing new therapeutic strategies that regard soluble mtDNA, as novel treatments could target either soluble MTDs, or the receptors they use. Thus, in HIV infection, as in other diseases characterized by excessive inflammation, interfering with MTDs could likely become a novel strategy to reduce the harmful immune activation.
2011
- Increased plasma levels of extracellular mitochondrial DNA during HIV infection: a new role for mitochondrial damage-associated molecular patterns during inflammation.
[Articolo su rivista]
Cossarizza, Andrea; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; Manzini, S; Roat, Erika; DE BIASI, Sara; Bertoncelli, Linda; Montagna, Jp; Bisi, Luca; Manzini, Lisa; Trenti, T; Borghi, V; Mussini, Cristina
abstract
HIV infection is characterized by a chronic inflammatory state. Recently, it has been shown that mitochondrial DNA (mtDNA) released from damaged or dead cells can bind Toll like receptor-9 (TLR9), an intracellular receptor that responds to bacterial or viral DNA molecules. The activation of TLR9 present within monocytes or neutrophils results in a potent inflammatory reaction, with the production of proinflammatory cytokines. We measured plasma levels of mtDNA in different groups of HIV(+) patients, i.e., those experiencing an acute HIV infection (AHI), long term non progressors (LTNP), late presenters (LP) taking antiretroviral therapy for the first time, and healthy controls. We found that in AHI and LP mtDNA plasma levels were significantly higher than in healthy individuals or in LTNP. Plasma mtDNA levels were not correlated to peripheral blood CD4(+) T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA in inflammation, or as a biomarker of virus-induced damage.
2011
- NK-cell phenotype at interruption underlies widely divergent duration of CD4+-guided antiretroviral treatment interruption.
[Articolo su rivista]
Bozzano, F; Nasi, Milena; Bertoncelli, Linda; Nemes, Elisa; Prati, F; Marras, F; Mussini, Cristina; Moretta, L; Cossarizza, Andrea; De Maria, A.
abstract
Long-term side effects may represent a relevant burden of antiretroviral treatment (ART) in HIV-infected patients with good CD4 immune reconstitution over extended time spans. CD4-guided treatment interruption (TI) has been evaluated to address this point and may result in a wide spectrum of time off ART in different patient cohorts. We studied whether differences in innate immune responses, in particular NK cells, are associated to patterns of longer (LoTI) or a shorter (ShTI) TI. Clinical cohort parameters were analyzed on a group of patients widely diverging for TI duration (<9 versus >18 months) on samples before TI, including NK-cell analysis and function by natural cytotoxicity receptor (NCR)-triggered γ-IFN production. Although persistently reduced NCR expression (NKp30) and function were observed in both LoTI and ShTI patients on ART compared with healthy donors, relevant differences were observed at baseline TI in those patients who subsequently developed LoTI course. Lower expression of NKG2D and NKp46 on NK cells. This also translates in reduced γ-IFN production in redirected functional assays. Thus, differences in innate immune balance exist during ART, may be associated to differential control of HIV infection and their understanding could explain clinical differences in individual patients that are not reflected by CD4(+) cell counts alone.
2011
- Quercetin and cancer chemoprevention
[Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Nasi, Milena; J. P., Montagna; DE BIASI, Sara; Roat, Erika; Bertoncelli, Linda; E. L., Cooper; Cossarizza, Andrea
abstract
Several molecules present in the diet, including flavonoids, can inhibit the growth of cancer cells with an ability to act as "chemopreventers". Their cancer-preventive effects have been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. The antioxidant activity of chemopreventers has recently received a great interest, essentially because oxidative stress participates in the initiation and progression of different pathological conditions, including cancer. Since antioxidants are capable of preventing oxidative damage, the wide use of natural food-derived antioxidants is receiving greater attention as potential anti-carcinogens. Among flavonoids, quercetin (Qu) is considered an excellent free-radical scavenging antioxidant, even if such an activity strongly depends on the intracellular availability of reduced glutathione. Apart from antioxidant activity, Qu also exerts a direct, pro-apoptotic effect in tumor cells, and can indeed block the growth of several human cancer cell lines at different phases of the cell cycle. Both these effects have been documented in a wide variety of cellular models as well as in animal models. The high toxicity exerted by Qu on cancer cells perfectly matches with the almost total absence of any damages for normal, non-transformed cells. In this review we discuss the molecular mechanisms that are based on the biological effects of Qu, and their relevance for human health.
2010
- Cytotoxic granule release dominates gag-specific CD4+ T-cell response in different phases of HIV infection.
[Articolo su rivista]
Nemes, Elisa; Bertoncelli, Linda; Lugli, Enrico; Pinti, Marcello; Nasi, Milena; Manzini, Lisa; Manzini, Simona; Prati, Francesca; Borghi, V; Cossarizza, Andrea; Mussini, Cristina
abstract
BACKGROUND: The activity of virus-specific T lymphocytes, among which those capable of a polyfunctional response against the viral protein gag, is crucial to control HIV infection. OBJECTIVE: The objective of this study is to investigate the polyfunctionality of gag-specific T cells in different phases of HIV infection, analyzing markers related to T-helper cell 1 (Th1) and degranulation/cytotoxicity, and the production of Th1 cytokines in peripheral blood lymphocytes from patients experiencing an acute primary infection, long-term nonprogressors, patients naive for antiretroviral drugs, and patients taking HAART. MATERIALS AND METHODS: Cells were stimulated with a pool of gag-derived peptides or with a superantigen (staphylococcal enterotoxin B). Using eight-color polychromatic flow cytometry, we analyzed the expression of interleukin-2, interferon-gamma, CD154, and CD107a by CD4 and CD8 T cells. RESULTS: The main finding was that in all HIV-positive patients, about half gag-specific CD4 T cells were CD107a, that is, able to degranulate. CD4CD154 cells unable to produce Th1 cytokines were the second most represented population. Truly polyfunctional CD4 T cells were very rare and present only in a few long-term nonprogressors. Superantigen stimulation showed that CD4 T lymphocytes from all patients displayed a typical Th response, including interleukin-2 and interferon-gamma production, lacking CD107a expression. CONCLUSION: In all the aforementioned phases of HIV infection, the large majority of gag-specific CD4 T lymphocytes cannot be identified by the sole expression of interleukin-2 and interferon-gamma, which is early impaired. Degranulation and helper functions other than Th1 cytokine production are the predominant features of HIV-specific CD4 lymphocytes.
2010
- Effect of antioxidants on mitochondrial function in HIV-1-related lipoatrophy: a pilot study.
[Articolo su rivista]
Milazzo, L; Menzaghi, B; Caramma, I; Nasi, Milena; Sangaletti, O; Cesari, M; Poma, Bz; Cossarizza, Andrea; Antinori, S; Galli, M.
abstract
We investigated the effect of antioxidant supplementation on mitochondrial function, fat distribution, and lipid and glucose metabolism in HIV-1-infected patients with antiretroviral therapy (ART)-related lipoatrophy. 61 ART-treated HIV-1-infected patients with lipoatrophy were randomized to receive either n-acetyl-L-carnitine (n = 21), lipoic acid + n-acetylcisteine (LA/NAC) (n = 20), or no supplementation (n = 20) for 48 weeks. At baseline and at the end of treatment, mitochondrial function was studied by (13)C-methionine breath test and by mitochondrial (mt)-DNA quantification on circulating T-cells and subcutaneous adipose tissue. Body composition was assessed by dual-energy X-ray absorpiometry (DEXA). (13)CO(2)-exhalation increased between baseline and week 48 in both supplementation arms as evidenced by a higher delta over baseline excretion at 45 min (from mean ± SEM of 7.8 ± 1.08 to 9.9 ± 0.6, p = 0.04 in the n-acetyl-carnitine arm, and from 7.4 ± 0.8 to 11.5 ± 1.6, p = 0.01 in LA/NAC arm). Cumulative (13)CO2 excretion increased from median (interquartile range; IQR) of 3.25 (2.55-4.2) to 4.51 (4.12-5.2) in the carnitine arm; from 3.79 (2.67-4.37) to 4.83 (4.25-5.56) in the LA/NAC arm; p = 0.004, 0.02, respectively. mtDNA content increased in CD4+ T-cells from patients who received n-acetyl-carnitine (+30 copies/cell; p = 0.03), without significant difference by the overall comparison of the study groups. Fat body mass and lipid profile did not change significantly in any of the arms. Our study showed that antioxidant supplementation may have a protective role on mitochondrial function, with limited effects on the reversal of clinical lipodystrophic abnormalities in HIV-1-infected patients.
2010
- Interfering with ROS metabolism in cancer cells: the potential role of quercetin.
[Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Nasi, Milena; DE BIASI, Sara; Roat, Erika; Bertoncelli, Linda; Cossarizza, Andrea
abstract
Abstract: A main feature of cancer cells, when compared to normal ones, is a persistent pro-oxidative state that leads to an intrinsic oxidative stress. Cancer cells have higher levels of reactive oxygen species (ROS) than normal cells, and ROS are, in turn, responsible for the maintenance of the cancer phenotype. Persistent ROS stress may induce adaptive stress responses, enabling cancer cells to survive with high levels of ROS and maintain cellular viability. However, excessive ROS levels render cancer cells highly susceptible to quercetin, one of the main dietary flavonoids. Quercetin depletes intracellular glutathione and increases intracellular ROS to a level that can cause cell death.
2010
- Mitochondrial changes during D-drug-containing once-daily therapy in HIV-positive treatment-naive patients.
[Articolo su rivista]
Maggiolo, F.; Roat, Erika; Pinti, Marcello; Nasi, Milena; Gibellini, Lara; DE BIASI, Sara; Airoldi, M.; Ravasio, V.; Mussini, Cristina; Suter, F.; Cossarizza, Andrea
abstract
BACKGROUND: Antiviral drugs of the category of nucleoside reverse transcriptase inhibitors (NRTIs), largely used for the treatment of HIV infection, can have toxic effects on mitochondria. We performed a cross-sectional study on mitochondrial toxicity in a randomized group of patients belonging to a larger randomized study on different NRTI-based once-daily regimens by quantifying mitochondrial DNA (mtDNA), three different mitochondrial RNAs (mtRNAs) and functional parameters in highly purified peripheral CD4+ and CD8+ T-cells.METHODS: A total of 49 previously treatment-naive patients treated for a mean of 15 months with efavirenz plus didanosine plus lamivudine (group 1), or tenofovir disoproxil fumarate plus lamivudine (group 2), or didanosine plus abacavir (group 3) were considered. The groups were matched for sex, age, CDC classification, risk factor for HIV, nadir CD4+ T-cell count and baseline viral load. mtDNA and mtRNA were quantified by using real-time PCR assays.RESULTS: No patient showed any clinical symptom; however, the amount of mtDNA in CD4+ and CD8+ T-cells was significantly lower in groups 1 and 3; similarly, the expression of different mtRNAs in both CD4+ and CD8+ T-cells showed significant differences that were dependent upon the drug used. No differences were found in mitochondrial membrane potential and mitochondrial mass in peripheral lymphocytes. The amount of total HIV DNA in CD4+ T-cells did not differ among the groups, who displayed a similar immune reconstitution and control of the virus.CONCLUSIONS: An efficient didanosine-containing once-daily therapy can have more mitochondrial toxicity than regimens devoid of this drug.
2010
- Predictive Value of Intracellular HIV-1 DNA Levels During CD4-Guided Treatment Interruption in HIV(+) Patients
[Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Manzini, S; Gibellini, Lara; Manzini, Lisa; Bisi, Luca; DE BIASI, Sara; DEL GIOVANE, Cinzia; D'Amico, Roberto; Borghi, V; Mussini, Cristina; Cossarizza, Andrea
abstract
The amount of HIV-1 DNA within peripheral blood mononuclear cells is an important marker of viral activity. We studied intracellular HIV-1 DNA content in purified CD4(+) T cells from 28 chronically HIV-1-infected adults with sustained CD4(+) T cell counts (>500 cells/microl) and undetectable plasma viral load (<50 copies/ml), who underwent CD4-guided treatment interruption (TI). Patients were followed up for 18 months during TI, and for 6 months after treatment resumption (TR). Six naïve HIV(+) patients starting therapy were also enrolled and followed up for 6 months. All patients were studied every 2 months; HIV-1 DNA copy number was quantified with real-time PCR. Considering all patients remaining off-treatment, in the first 18 months of TI, intracellular HIV-1 DNA levels (expressed as Log(10) copies/million cells) remained stable (mean, 3.82 and 3.77 at time 0 and after 18 months, respectively). Similarly, HIV-1 DNA values, either in patients who restarted treatment after TI (time 0, 4.90) or in naïve patients who started treatment for the first time (time 0, 4.37), did not change significantly in the first 6 months of therapy (4.42 and 3.67, respectively). Evaluating HIV-1 DNA variations during the first 2 months of TI, we found that patients with a stable level had a lower risk to reach a CD4(+) T cell count <350 cells/microl, and thus to restart therapy, whereas this risk was significantly higher in those with a marked increase of HIV-1 DNA. In conclusion, intracellular HIV-1 DNA is a predictive marker for the length of CD4-guided TI.
2010
- T cell homeostasis in centenarians: from the thymus to the periphery.
[Articolo su rivista]
Pinti, Marcello; Nasi, Milena; Lugli, Enrico; Gibellini, Lara; Bertoncelli, Linda; Roat, Erika; DE BIASI, Sara; Mussini, Cristina; Cossarizza, Andrea
abstract
The immune system undergoes a process of profound remodelling during aging, referred to as immunosenescence, and characterized by complex modifications of several components. In this review, we discuss recent developments and observations regarding the generation of T cells in the thymus during aging and longevity, and the regulation and maintenance of peripheral blood lymphocytes. The generation of new T cells is indeed crucial to maintain a functional immune system, and is a fundamental step to avoid unsuccessful aging, thus reaching longevity in good health. Mechanisms will be described that are related to the production and maintenance of those lymphocytes defined "recent thymic emigrants", and to the detection of the so called "T cell receptor rearrangement excision circles (TREC)", along with the presence in the periphery of naïve and memory T cells, that can be influenced and regulated by several different mechanisms. Several strategies aimed at improving thymic functionality are currently receiving a growing interest, and some of them are based on molecules that are produced by, and/or act on immune cells. Data on the possible use of these molecules, including cytokines like interleukin (IL)-7, IL-15 and keratinocyte growth factor, to restore thymic function are reviewed and discussed.
2010
- The Role of Mitochondria in HIV Infection and Its Treatment
[Articolo su rivista]
Pinti, M.; Nasi, M.; Gibellini, L.; Roat, E.; De Biasi, S.; Bertoncelli, L.; Cossarizza, A.
abstract
Mitochondria play a dual role in the life of the cell, being capable of producing either energy (in the form of ATP) or potentially dangerous reactive oxygen species (ROS), and they also contain molecules that, when released into the cytoplasm, cause apoptosis. There is a growing interest in the importance of these organelles during the infection caused by the human immunodeficiency virus (HIV), as well as during its treatment. Indeed, several drugs that are capable of blocking HIV can also interact with the enzyme responsible for the replication of mitochondrial DNA and inhibit its activity. Cytokines produced by the immune system can alter ROS production. Furthermore, the virus as such can trigger different mechanisms that interfere with mitochondrial functionality and induce alterations, ultimately causing cell death. As a result, mitochondria can be severely altered by HIV infection and by its treatment. © 2010 Taiwan Medical University.
2010
- Upregulation of nuclear-encoded mitochondrial LON protease in HAART-treated HIV-positive patients with lipodystrophy: implications for the pathogenesis of the disease
[Articolo su rivista]
Pinti, Marcello; Gibellini, Lara; Guaraldi, Giovanni; Orlando, Gabriella; Gant, Tw; Morselli, Eugenia; Nasi, Milena; Salomoni, Paolo; Mussini, Cristina; Cossarizza, Andrea
abstract
BACKGROUND: HAART can provoke metabolic changes and body fat redistribution, resulting in lipodystrophy, a side effect significantly involving mitochondrial function. Mitochondrial DNA (mtDNA) depletion caused by nucleosidic reverse transcription inhibitors is supposed to be a crucial mechanism in the pathogenesis of mitochondrial damages. METHODS: In adipose tissue from 22 HIV-positive patients with lipodystrophy and 20 healthy controls, we analyzed gene expression by microarray analysis and real-time PCR. The most upregulated gene was further studied in the human adipocytic cell line SW872 by real-time PCR, western blot, transient transfection assays and flow cytometry. RESULTS: We identified 18 genes differently expressed between lipodystrophy patients and controls, and focused our attention on the nuclear-encoded mitochondrial protease LON, essential in mtDNA maintenance. In SW872 cells, treatment with stavudine (d4T) doubled LON levels, in parallel with mtDNA depletion. As d4T increased reactive oxygen species (ROS) intracellular content, we measured LON in presence of deoxyribose, which causes oxidative stress but not mtDNA depletion, and observed LON upregulation. Ethidium bromide, which markedly depletes mtDNA, did not alter LON levels. The antioxidant glutathione inhibited the increase of intracellular ROS and the increase in LON caused by d4T or deoxyribose. CONCLUSION: LON upregulation was due to d4T-induced ROS production, rather than due to mtDNA depletion, and represents a response to an oxidative stress. Other mechanisms than mtDNA depletion thus exist that explain nucleosidic reverse transcription inhibitors toxicity. This observation provides a rationale for possible therapeutic interventions aimed at reducing intracellular ROS content in patients assuming HAART.
2009
- Lymphocytes sub-types and functions in centenarians as models for successful ageing
[Capitolo/Saggio]
Lugli, E.; Troiano, L.; Pinti, M.; Nasi, M.; Roat, E.; Ferraresi, R.; Bertoncelli, L.; Gibellini, L.; Nemes, E.; Cossarizza, A.
abstract
Several cell subsets participate to the immune response, and their close interplay is fundamental for the successful elimination of harmful pathogens. In addition, a tight regulation of the immune response has to occur in order to avoid excessive inflammation and potential autoreactivity towards self components. In the last years, the discovery and the characterization of new lymphocytes subsets, including regulatory T (Treg)-cells and Natural Killer T (NKT)-cells allowed a better understanding of how an effector immune response is induced and therefore down-modulated. During the ageing of the immune system, a process termed immunosenescence, these subsets undergo a profound remodelling, both in phenotype and function. In this chapter, we will describe the essential features of lymphocyte populations in centenarians and the differences that occur with unsuccessfully aged people.
2009
- Quercetin inhibits lymphocyte activation and proliferation without inducing apoptosis in peripheral mononuclear cells.
[Articolo su rivista]
Lugli, Enrico; Ferraresi, Roberta; Roat, Erika; Troiano, Leonarda; Pinti, Marcello; Nasi, Milena; Nemes, Elisa; Bertoncelli, L; Gibellini, Lara; Salomoni, Paolo; Cooper, El; Cossarizza, Andrea
abstract
Toxicity of chemotherapeutic drugs towards normal cells is a serious side effect of cancer treatment. Thus, finding of molecules with low toxicity for normal cells is crucial. Several natural compounds, such as flavonoid quercertin, are receiving a growing attention as “chemopreventers”. Quercetin kills tumour-derived cell lines, but little is known about its effects on normal cells. Here we show that although quercetin exerts a higher apoptotic potential on leukemic cell lines than on peripheral blood mononuclear cells (PBMCs) and does not sensitize PBMCs to CD95-induced apoptosis, it is able to inhibit normal immune functions such as T cell proliferation and activation. Quercetin sensitivity is independent on cell cycle progression since it was not abrogated in serum-starved U937 cells, nor proliferating PBMCs underwent apoptosis after quercetin treatment. However, quercetin prevented PHA-induced PBMC proliferation and SEB-induced upregulation of activation markers. Our data suggest that quercetin, while incapable of inducing apoptosis in normal cells under several conditions, could interfere with effector T cell function.
2009
- Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry
[Articolo su rivista]
Cossarizza, Andrea; Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; Gibellini, Lara; Bertoncelli, Linda; Nasi, Milena; Pinti, Marcello
abstract
Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death.
2008
- Effects of the change from Stavudine to tenofovir in human immunodeficiency virus-infected children treated with highly active antiretroviral therapy: studies on mitochondrial toxicity and thymic function.
[Articolo su rivista]
Rosso, Sabina; Nasi, Milena; Di Biagio, A.; Repetto, E.; Dentone, C.; Pinti, Marcello; Nemes, Elisa; Ferraresi, Roberta; Mussini, Cristina; Esposito, Roberto; Viscoli, C.; Cossarizza, Andrea
abstract
BACKGROUND:Changing from drugs that have significant mitochondrial toxicity to less toxic compounds may be of benefit in human immunodeficiency virus (HIV)-positive patients who receive highly active antiretroviral therapy. Few data on mitochondrial toxicity of antiviral drugs are available in HIV-positive children. METHODS: Eighteen HIV-positive children (median age, 10.9 years) receiving a stavudine-containing regimen were randomized to maintain stavudine (arm A) or change to tenofovir (arm B), while preserving the remaining drugs. Glucose, lipidic, and viro-immunologic factors were assessed at months 0, 1, 3, 6, 12, and 18. Thymic output and mtDNA content were measured in peripheral blood mononuclear cells at 0 and 6 months, mtDNA in isolated CD4+ and CD8+ T cells after 18 months. RESULTS: From baseline to month 6, arms A and B showed similar thymic output and mtDNA. After 18 months, a significant decrease in plasma HDL was observed in arm B, along with a small increase in blood glucose; mtDNA showed no difference. In the 2 arms other factors did not show significant differences from the baseline and from the previous values at 18 months. CONCLUSIONS: Changing from stavudine to tenofovir was well-tolerated, and viro-immunologic success was maintained.
2008
- Genetic polymorphisms differently influencing the emergence of atrophy and fat accumulation in HIV-related lipodystrophy
[Articolo su rivista]
Zanone Poma, B.; Riva, A.; Nasi, Milena; Cicconi, P.; Broggini, V.; Cozzi Lepri, A.; Mologni, D.; Mazzotta, F.; D’Arminio Monforte, A.; Mussini, Cristina; Cossarizza, Andrea; Galli, M.
abstract
Objective and design: The present study aims at evaluating the influence of geneticpolymorphisms on antiretroviral therapy (ART)-associated lipodystrophy. We includedin the study 255 ICoNA. patients and we assessed the distribution of Fas 670 AGpolymorphism, ApoC3 455 CT and 482 CT polymorphisms, C161T silent substitutionin the PPAR g gene, the Adrenergic b3 Receptor (ARb3) codon 64 TC variant, andtwo polymorphisms in the Adrenergic b2 Receptor (ARb2) codon 16 AG and codon 27CG. Crude rates of lipoatrophy and fat accumulation and adjusted relative rates werecalculated using Poisson regression.Results: In a multivariate model after adjusting for gender, HIV exposure, age, currentviral load, hepatitis C virus (HCV) serology, nucleoside reverse-transcriptase inhibitor(NRTI) pair/‘third drugs’ currently used, months of pre-highly active antiretroviraltherapy (HAART) exposures to NRTI, the following genotypes resulted protectiveagainst lipoatrophy: ApoC3 455 CC genotype [adjusted relative risks (ARR) 0.2,95% confidence interval (CI) 0.046–0.91 vs CT/TT, P¼0.037], ARb3 codon 64 TTgenotype (ARR 0.39, 95% CI 0.14–1.06 vs TC/CC, P¼0.066), and Fas 670 GGgenotype (ARR 0.51, 95% CI 0.26–1.01 vs AG/AA, P¼0.053). With regard to fataccumulation, in the multivariate model, the ARb2 codon 27 CC genotype resultedprotective (ARR 0.21, 95% CI 0.08–0.51 vs CG/GG, P¼0.0006), whereas the ARb2codon 16 AA genotype resulted associated with higher risk (ARR 3.72, 95% CI 1.58–8.76 vs AG/GG, P¼0.0026).Conclusion: Our study suggests that genetic polymorphisms of genes involved inapoptosis and adipocyte metabolism are significantly related to ART associated lipodystrophy.Particularly, we evidenced a role for ApoC3 455 in lipoatrophy and for thetwo variants of ARb2 in fat accumulation.
2008
- Homeostatic cytokines and expansion of regulatory T cells accompany thymic impairment in children with Down syndrome
[Articolo su rivista]
Roat, E; Prada, N; Lugli, E; Nasi, Milena; Ferraresi, R; Troiano, L; Giovenzana, C; Pinti, Marcello; Biagioni, O; Mariotti, M; Di Iorio, A; Consolo, Ugo; Balli, F; Cossarizza, Andrea
abstract
Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the alpha chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels of IL-7 and IL-15 than in healthy controls, and a higher proliferative T cell response to IL-15. DS children also showed a lower percentage of CD4(+) cells and profound alterations of T cell differentiation, along with increased amount of Tregs and of cells expressing markers of apoptosis. We can thus hypothesize that the precocious thymic involution occurring in DS is mirrored by a high production of IL-7 and IL-15, which is crucial for cell survival and proliferation. The complex alterations present in the periphery are likely the result of a compensatory mechanism: the overproduction of homeostatic cytokines could be a reaction to the impaired intrathymic production of T lymphocytes and/or to the expansion of Treg in the periphery, and could be required to allow the survival of T cells.
2008
- Mitochondrial DNA Haplogroups and Highly Active Antiretroviral Therapy–Related Lipodystrophy
[Articolo su rivista]
Nasi, Milena; Guaraldi, Giovanni; Gabriella, Orlando; Caterina, Durante; Pinti, Marcello; Elisa, Nemes; Nardini, Giulia; Giuseppe, Passarino; Cocchi, Marina; Esposito, Roberto; Mussini, Cristina; Cossarizza, Andrea
abstract
Background. The combination of different point mutations in mitochondrial DNA (mtDNA), which are defined as haplogroups, may cause modification in organelle function and may be involved in several pathologies. We analyzed the distribution of mtDNA polymorphisms in human immunodeficiency virus (HIV)–infected patients with lipodystrophy, a relevant adverse event caused by highly active antiretroviral therapy, and their correlation with metabolic and viroimmunologic parameters. Methods. The frequency of the 9 most common European haplogroups was investigated in 346 white, HIV- infected patients with lipodystrophy. Haplogroups were identified on the basis of classic methods. Statistical analysis was performed with use of 1-way analysis of variance, the x2 test, and principal-components analysis. Results. The distribution of mtDNA haplogroups among patients with lipodystrophy was similar to that among the general European population. We found no differences between patients with different haplogroups with regard to viroimmunologic results (plasma HIV load, CD4+ T cell count, and nadir CD4+ T cell count), glucose data (glucose, insulin, C-peptide, and glycosylated hemoglobin concentrations and insulin resistance), lipid data (levels of triglycerides, total cholesterol, high- and low-density lipoproteins, and apolipoprotein A1 and B), acid-base balance parameters (lactate level and anion gap), or anthropometric measures (weight, body mass index, and waist- to-hip ratio). No differences were observed in trunk fat levels, leg-fat ratio (which was determined by dual-energy X-ray absorptiometry), or exposure to different drug classes. Principal-components analysis confirmed that the spatial distribution of patients belonging to a given haplogroup was not influenced by different clinical parameters. Conclusions. Our study indicates that, in HIV-infected patients with lipodystrophy, mtDNA haplogroups are not related to major metabolic changes or to particular viroimmunologic features.
2008
- Physiology and immunology of the thymus gland
[Capitolo/Saggio]
Nasi, Milena; Pinti, Marcello; Troiano, Leonarda; Cossarizza, Andrea
abstract
The thymus is a giand located in the upper anteriorportion of the chest cavity just behind the sternurn,Under the evolutionary pressure exerted by the emergence of adaptive immunityAnd its inherent risk to from receptorsThat recognize self molecules, this gland appeared about 500 milT cells in order to prevent autoimmunity and orchestrate selftolerance.The thymus has thus become a crucial lymphoid organ in which cells arriving from the bone marrow undergo a finely tuned process of selection based on the specificity of T-cell receptors (TCRs) and differentiate into mature T-cells.The development of thymocitesn involves a stringent selection in which only 1-3% of these cells succeed in survival and can leave the gland to colonize the periphery and give origin to effective immune cells. Duting maturation in thymus, T cells are first positively selected for uselfulness and then negatively selected against autoreactivity. These intrathymis events are governed by sequential interactions of thymocites with different stromal cell types during the migration through the thymus essentially from the external to the internal part of the thymic lobuli. The mature T cells called naïve T cells leave the organ and contribute to the peripheral T cell poll. The complex process of intrathymic maturation of T-lymphocites involvesvarious thymic specific factors and several other molecules. Indeed T cell maturation requires either direct cell-to-cell or paracrine interactions that occur via cytokines or thymic hormones produced by the cells of the thymic microenvironment. For a long toime the functions of the thymus have remained obscure. The first demonstration of its crucial role in the ontogeny and development of the immune system was provided in 1961 when it was shown that mice thymectomized immediately after birth had poorly developed lymphoid tisses impaired immune responses and susceptibility to infections. Althought cells present in the thymus were believed to be immunoincompetent a few years later it was shown that they could proliferate after an antigenic challenge and produce cells unable to synthetize antibodies. Such cells were capable of enabling other lymphocyte to differentiate to antibody-forming cells. This was the first demonstration in mammalians of the existence of two major subsets of lymphocytes now known as T- and B-cells. It required a re-evaluation of many immunological phenomena such as tolerance memory and autoimmunity and it was followed by a huge number of studies elucidating many of the mysteries of the immune system.
2008
- Plasma HIV load and proviral DNA decreases after two standard antiretroviral regimens in HIV-positive patients naïve to antiretrovirals
[Articolo su rivista]
Torti, C; Quiros Roldan, Me; Cologni, G; Nichelatti, M; Ceresoli, F; Pinti, Marcello; Nasi, Milena; Cossarizza, Andrea; Lapadula, G; Costarelli, S; Manca, N; Gargiulo, F; Magoni, M; Carosi, G.
abstract
To compare early decrease of HIV plasma viral load (pVL) after two standard combinations of highly active antiretroviral therapy (HAART). (ii) To evaluate variations of proviral HIV-DNA load on conditions of sustained pVL undetectability. Two different sub-studies of a multicentre prospective randomized controlled trial which compared two first-line HAART (i.e., zidovudine+lamivudine+lopinavir/ritonavir versus tenofovir+lamivudine+ efavirenz). Only patients enrolled at the coordinating centre (University of Brescia) were included in the two sub-studies. In the first sub-study, we calculated pVL decrease with respect to baseline at any of the following time-points: days 1, 3, 7, 14 and 28. Decreases of the pVL were compared between the two treatment groups. In the second sub-study, we analyzed variation of proviral HIV-DNA load in CD4+ T-cells from baseline to week 52 only in patients who maintained the same treatment regimen and had sustained undetectable pVL. In either studies, linear regression analysis was used to investigate what factors could influence variations of pVL and of proviral HIV-DNA load. (i) 64 patients were studied. A significant decrease of pVL was found from day 3 on, without statistically significant differences between the two study groups. However, after adjusting for possible confounders, tenofovir+lamivudine+efavirenz resulted to be associated with greater pVL decreases. (ii) 45 patients were studied. Mean proviral HIV-DNA load decreased from 1,610 (95%CI: 879-2,341) to 896 (95% CI 499-1,293) copies/10(6) cells (P=0.05). Linear regression analysis showed that the decrease of proviral DNA load during follow-up was independently and inversely correlated with age. Further studies are needed to compare pVL decay between antiretroviral regimens and assess whether proviral HIV-DNA load is a surrogate marker of treatment effectiveness.
2007
- Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome.
[Articolo su rivista]
Roat, Erika; Prada, Nicole; Ferraresi, Roberta; Giovenzana, Chiara; Nasi, Milena; Troiano, Leonarda; Nemes, Elisa; Pinti, Marcello; Lugli, Enrico; O., Biagioni; M., Mariotti; L., Ciacci; Consolo, Ugo; Balli, Fiorella; Cossarizza, Andrea
abstract
Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (Delta Psi(m)) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of Delta Psi(m), underlying the presence of an increasing susceptibility of these organelles to damaging agents. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.</div>
2007
- Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.
[Articolo su rivista]
Troiano, Leonarda; Ferraresi, Roberta; Lugli, Enrico; Nemes, Elisa; Roat, Erika; Nasi, Milena; Pinti, Marcello; Cossarizza, Andrea
abstract
The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.
2007
- Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data
[Articolo su rivista]
Lugli, Enrico; Pinti, Marcello; Nasi, Milena; Troiano, Leonarda; Ferraresi, Roberta; Mussi, Chiara; Salvioli, Gianfranco; Patsekin, V; Robinson, Jp; Durante, Caterina; Cocchi, Marina; Cossarizza, Andrea
abstract
BACKGROUND: Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification. METHODS: By eight-colour cytofluorimetric analysis, we investigated the T cell compartment in donors of different age (young, middle-aged, and centenarians). T cell subsets were identified by combining positive and negative expression of antigens. The resulting data set was organized into a matrix and subjected to CA and PCA. RESULTS: CA clustered people of different ages on the basis of cytofluorimetric profile. PCA of the cellular subsets identified centenarians within a different cluster from young donors, while middle-aged donors were scattered between these groups. These approaches identified T cell phenotypes that changed with increasing age. In young donors, memory T cell subsets tended to be CD127+ and CD95- whereas CD127-, CD95+ phenotypes were found at higher frequencies in people with advanced age. CONCLUSIONS: Our data suggest the use of bioinformatic approaches to analyze large data-sets generated by PFC and to obtain the rapid identification of key populations that best characterize a group of subjects. (c) 2007 International Society for Analytical Cytology.
2006
- Immunophenotype of HIV plus patients during CD4 cell-monitored treatment interruption: role of the IL-7/IL-7 receptor system
[Articolo su rivista]
Nemes, E.; Lugli, E.; Nasi, M.; Ferraresi, R.; Pinti, M.; Bugarini, R.; Borghi, V.; Prati, F.; Esposito, R.; Cossarizza, A.; Mussini, C.
abstract
Objective: To investigate immunological changes during CD4-guided therapy interruption in HIV+ patients who suspended HAART. Patients: Seventeen patients aged > 18 years, who had received HAART for at least 12 months, and had a pre-interruption CD4+ cell count > 500 cells/mu d, interrupted treatment. Median nadir CD4+ cell count was 288 cells/mu l. HIV plasma viral load at discontinuation was < 50 or > 50 copies/ml. Criteria for restarting treatment were: a CD4+ T-lymphocyte count < 350 cells/ d on two separate occasions, a clinical manifestation of AIDS, and the patient's desire to resume HAART. Eleven patients were still off therapy after 12 months (group A); according to the first criterion, six patients restarted therapy within 12 months (group B). Methods: Haernatological, viro-immunological, cytofluorimetic and molecular assays were performed at baseline and every 2 months following standard methods. Statistical analysis was performed under Stata 7.0. Results: In the first 2 months of treatment interruption, a significant increase in viral load and CD8+ lymphocyte activation occurred. Then such parameters decreased and remained stable. In all patients, a decrease in CD4+ lymphocytes took place as well, that affected in a similar manner naive, central memory, effector memory and terminally differentiated cells. Group B always presented lower amounts of CD4+ effector memory lymphocytes. The expression of CD`127 was always higher in group A. Conclusions: The loss of CD4+ lymphocytes upon viral rebound is equal among naive and memory subsets. Patients with higher expression of CD127, who are likely to exert a better capacity to utilize endogenous interleukin-7 by T cells, could remain off therapy for longer periods. (c) 2006 Lippincott Williams & Wilkins
2006
- Modulation of CD38 expression in human longevity: A flow cytometric study
[Abstract in Atti di Convegno]
Lugli, Enrico; Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; Ferraresi, Roberta; Roat, Erika; Durante, Caterina; Cocchi, Marina; Cossarizza, Andrea
abstract
The dynamics of CD38 expression innewborns and young healthy donors hasbeen widely investigated for many years.However, little is known about the modulationof this marker during humanageing. We analyzed the changes inCD38 expression in peripheral bloodlymphocytes (PBL) from subjects whowere centenarians. For this purpose weused polychromatic flow cytometry, apowerful technology that allows the determinationof multiple antigens (in ourcase, up to 8) present in the same cell.Among the subsets within CD4+ andCD8+ T cell populations identified bythis approach, we investigated the expressionof CD38 together with markersrelated to extrathymic T cell differentiation(CD45RA and CCR7), T cell survival(CD127/IL-7rα) and activation/apoptosis(CD95). The groups analysed includedyoung donors (21±2 years old),middle-aged individuals (60±1.5 yearsold) and centenarians.By automatic boolean gating, we identifiedall the possible subsets obtained bythe combination of positive and negativeexpression for each marker indicatedabove. Moreover, we could distinguish betweendim or bright expression of CD38.CD38 expressed by CD4+ T cells doesnot show significant modifications in thethree samples either in of the virgin ormemory subsets.A slight increase in CD38 expressionwas found in PBL CD8+ T cells from centenarians.These CD8+/38dim T cells displayeda CD45RA-/CCR7+ central memoryor CD45RA-/CCR7- effector memoryphenotype. Further, CD38 expressionwas associated with the presence ofCD95 and the absence of CD127/IL-7rα.These results were also confirmed byCluster Analysis (CA) and PrincipalComponent Analysis (PCA) of the highnumber of T cell populations identifiedby flow cytometry. These bioinformatictechniques cluster the individuals accordingto the flow cytometric profile,which confirmed that the subsets with anincreased expression of CD38 (CD38bright) are more frequent in the sampleof centenarians.In conclusion, our data indicate amodulation of CD38 expression in CD8+T cells during human ageing. In particular,the preferential coexpression of thisantigen with CD95 but not CD127/IL-7r_suggests an age-dependent acquisition ofan effector phenotype of CD8+ T cellswhich could, at least in part, explain thechronic pro-inflammatory status presentin centenarians.
2006
- Polymorphisms of Fas gene: Relationship with Alzheimer's disease and cognitive decline
[Articolo su rivista]
M., Chiappelli; Nasi, Milena; Cossarizza, Andrea; E., Porcellini; E., Tumini; Troiano, Leonarda; Pinti, Marcello; M., Franceschi; F., Licastro
abstract
The Fas antigen (CD95) is a cell surface receptor that mediates cell apoptosis signalling. Recent investigations have shown that Fas-regulated apoptosis was linked to neurodegenerative lesions in the brain of patients with Alzheimer's disease ( AD). Here data regarding the association of two polymorphisms of the Fas promoter region with AD patient's cognitive deterioration are reported. The polymorphism at position - 1377 was associated with the risk of developing AD and with a differential rate of cognitive decline during a 2-year follow-up. The polymorphism at position - 670 was not associated with the risk of AD and with the cognitive decline during the follow-up. Our data suggest that different genetic background in the Fas gene may influence the risk and clinical progression of the disease by affecting neurodegenerative processes leading to neuronal loss. Copyright (C) 2006 S. Karger AG, Basel.
2006
- Protective effect of acetyl-L-carnitine against oxidative stress induced by antiretroviral drugs
[Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; Nemes, Elisa; Lugli, Enrico; Nasi, Milena; Pinti, Marcello; M., Calvani; M., Iannuccelli; Cossarizza, Andrea
abstract
Both HIV infection per se and antiretroviral drugs might contribute to oxidative stress and mitochondrial dysfunctions. In this study we assess zidovudine, stavudine and didanosine on U937 and CEM cell lines. All these drugs induced apoptosis and increased intracellular hydrogen peroxide but not superoxide anions. The addition of acetyl-L-carnitine (ALC) was able to prevent the pro-oxidant effect of the drugs tested. Supplementation with ALC, deficient in certain cohorts of HIV-infected individuals, especially on high active antiretroviral therapy regimen, has been associated with favourable effects. These data suggest that one of these effects could be a direct anti-oxidant action. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
2006
- Thymic output and functionality of the IL-7/IL-7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life
[Articolo su rivista]
Nasi, Milena; Troiano, Leonarda; E., Lugli; Pinti, Marcello; Ferraresi, Roberta; Monterastelli, Elena; Mussi, Chiara; Salvioli, Gianfranco; Franceschi, Claudio; Cossarizza, Andrea
abstract
During aging, the thymus undergoes a marked involution that is responsible for profound changes in the T-cell compartment. To investigate the capacity of the thymus to produce new cells at the limit of human lifespan, we analyzed some basic mechanisms responsible for the renewal and maintenance of peripheral T lymphocytes in 44 centenarians. Thymic functionality was analyzed by the quantification of cells presenting the T-cell receptor rearrangement excision circles (TREC). A new method based upon real-time PCR was used, and we found that most centenarians (84%) had undetectable levels of TREC+ cells. Six-color cytofluorimetric analysis revealed that centenarians had an extremely low number of naive T cells; central memory and effector memory T cells were greatly increased, while terminally differentiated cells were as numerous as in young (aged 20-45) or middle-aged (aged 58-62) donors. Interleukin (IL)-7 and IL-7 receptor alpha-chain (CD127) levels were the same at all ages, as shown by ELISA, flow cytometry and real-time PCR. However, IL-7 plasma levels were higher in centenarian females than males. The presence of TREC+ cells and of very few naive T lymphocytes suggests that in centenarians such cells could either derive from residues of thymic lymphopoietic islets, or even represent long-living lymphocytes that have not yet encountered their antigen. IL-7 could be one of the components responsible, among others, for the higher probability of reaching extreme ages typical of females.
2005
- Altered mithocondrial RNA production in adipocytes from HIV-infected individuals with lipodystrophy
[Abstract in Rivista]
Galluzzi, L.; Pinti, Marcello; Guaraldi, Giovanni; Mussini, Cristina; Troiano, L.; Roat, E.; Giovenzana, C.; Nemes, E.; Nasi, M.; Orlando, G.; Salomoni, P.; Cossarizza, Andrea
abstract
Damage to mitochondria (mt) is a major side effect of highly active antiretroviral therapy (HAART) tha includes a nucleoside reverse transcriptase inhibitor (NRTI).Such damage is associated with the onset of lipodystrophy in HAART-treated HIV+ patients. To further investigate mt changes during this syndrome, we analysed the expression of mtRNA in adipocytes from lipodystrophic HIV+ patients taking NRTI-containing HAART and compared it with similar cells from healthy individuals.
2005
- Altered mitochondrial RNA production in adipocytes from HIV-infected individuals with lipodystrophy
[Articolo su rivista]
Galluzzi, Lorenzo; Pinti, Marcello; Guaraldi, Giovanni; Mussini, Cristina; Troiano, Leonarda; Roat, Erika; Giovenzana, Chiara; Nemes, Elisa; Nasi, Milena; Orlando, Gabriella; Salomoni, P; Cossarizza, Andrea
abstract
Background: Damage to mitochondria (mt) is a major side effect of highly active antiretroviral therapy (HAART) that includes a nucleoside reverse transcriptase inhibitor (NRTI). Such damage is associated with the onset of lipodystrophy in HAART-treated HIV+ patients. To further investigate mt changes during this syndrome, we analysed the expression of mtRNA in adipocytes from lipodystrophic HIV+ patients taking NIRTI-containing HAART and compared it with similar cells from healthy individuals. Materials and methods: Total RNA was extracted from adipocytes collected from different anatomical locations of 11 HIV+ lipodystrophic patients and seven healthy control individuals. RNA was reverse transcribed and Taqman-based real-time PCR was used to quantify three different mt transcripts (ND1, CYTB and ND6 gene products). mtRNA content was normalized versus the housekeeping transcript L13. Results: ND1, CYTB and ND6 expression was significantly reduced in HIV+ lipodystrophic patients. HIV+ men and women did not differ in a statistically significant way regarding the levels of ND1 and ND6, whereas the opposite occurred for CYTB. Conclusions: Lipodystrophy following treatment with NRTI-containing HAART is associated with a decrease in adipose tissue mtRNAs.
2005
- Changes in mitochondrial RNA production in cells treated with nucleoside analogues.
[Articolo su rivista]
Galluzzi, Lorenzo; Pinti, Marcello; Troiano, Leonarda; Prada, Nicole; Nasi, Milena; Ferraresi, Roberta; Salomoni, P; Mussini, Cristina; Cossarizza, Andrea
abstract
BACKGROUND: To investigate mitochondrial (mt) toxicity of antiretroviral drugs further, we developed a novel real-time PCR-based assay for the quantification of mtRNA. We analysed the effects of stavudine (d4T), didanosine (ddl) and zidovudine (AZT) on the production of mtRNAs in different human cell lines and compared the production with the amount of mtDNA present in the same cells. MATERIALS AND METHODS: HUT78, CEM and U937 cells were exposed to different nucleoside reverse transcriptase inhibitors (NRTIs) for 7 days. Thereafter, nucleic acids were isolated and Taqman-based real-time PCR was used to quantify mtDNA and three different mtRNAs (ND1, CYTB and ND6 gene products). RESULTS: Different amounts of mtRNAs exist in different cell lines. When mtRNA was measured in cells exposed to an NRTI, a marked decrease was observed in cells treated with d4T, but not with ddl or AZT. Changes in mtRNA production did not always correspond to modifications in mtDNA content: 1 microM d4T significantly changed mtRNA but not mtDNA content. CONCLUSIONS: d4T, but not ddl or AZT, significantly alters mtRNA quantity and quality. The method we have developed can reveal changes that are not observed by measuring mtDNA content only, and can be used for ex vivo studies on drug toxicity.
2005
- Characterization of cells with different mitochondrial membrane potential during apoptosis
[Articolo su rivista]
E., Lugli; Troiano, Leonarda; Ferraresi, Roberta; Roat, Erika; Prada, Nicole; Nasi, Milena; Pinti, Marcello; El, Cooper; Cossarizza, Andrea
abstract
Background: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (Delta Psi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different Delta Psi during apoptosis. Methods: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect AT, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass. Results: Treatment with Qu provoked the onset of three cell populations with different Delta Psi: (1) healthy cells, with normal Delta Psi, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate Delta Psi and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed Delta Psi that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate Delta Psi, were observed in other models of apoptosis. Conclusions: During Qu-induced apoptosis, loss of Delta Psi, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different Delta Psi, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound.
2005
- Direct analysis of thymic function in children with Down's syndrome.
[Articolo su rivista]
Nicole, Prada; Nasi, Milena; Troiano, Leonarda; Erika, Roat; Pinti, Marcello; Elisa, Nemes; Enrico, Lugli; Roberta, Ferraresi; Ciacci, Luigi; Davide, Bertoni; Ornella, Biagioni; Milena, Gibertoni; Cristina, Cornia; Liviana, Meschiari; Elisabetta, Gramazio; Mauro, Mariotti; Consolo, Ugo; Balli, Fiorella; Cossarizza, Andrea
abstract
BACKGROUND: Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS. METHODS: We studied 8 children affected by DS, aged 2-7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC). RESULTS: In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found. CONCLUSIONS: The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.
2005
- Effect of treatment interruption monitored by CD4 cell count on mitochondrial DNA content in HIV-infected patients: a prospective study
[Articolo su rivista]
Mussini, Cristina; Pinti, Marcello; R., Bugarini; V., Borghi; Nasi, Milena; Nemes, Elisa; Troiano, Leonarda; Guaraldi, Giovanni; Bedini, Andrea; C., Sabin; Esposito, Roberto; Cossarizza, Andrea
abstract
Background: HIV infection per se and HAART can alter mitochondrial functionality, leading to a decrease in mitochondrial DNA content. Objective: To evaluate whether treatment interruption monitored by CD4 cell count can restore mitochondrial DNA content in peripheral blood lymphocytes. Methods: Mitochondrial DNA content was measured in platelet-free CD4 and CD8 T cells by real-time polymerase chain reaction; flow cytometry was used to identify and quantify activated CD4 and CD8 T lymphocytes. Results: The 30 patients had been treated for a mean of 107 months (range, 27-197). Median CD4 cell count at discontinuation was 702 cells/mu l (range, 547-798). Median observational time from HAART discontinuation was 11.3 months (range, 4-26). Discontinuation of treatment provoked significant increases in mitochondrial DNA in CD8 T cells, which started only 6 months after therapy cliscontinuation [5.12 copies/ cell per month from 0 to 6 months (P = 0.3 7) and 2 6.96 copies/cel I per month from 6 to 12 months (P < 0.0001)]. Conclusions: This study is the first showing that mitochondrial DNA content can increase in peripheral blood lymphocytes during treatment interruption, but only after at least 6 months of interruption. Consequently, interruptions of shorter periods, whether by clinician or patient decision, are unlikely to allow restoration of mitochondrial DNA and so decrease HAART-related toxicity.
2005
- Effects on mitochondria and thymus of the switch from stavudine to tenofovir in HIV-infected children
[Abstract in Atti di Convegno]
Rosso, R; Nasi, Milena; Pinti, Marcello; Nemes, Elisa; Roat, Erika; Di Biagio, A; Repetto, E; Bassetti, M; Cossarizza, Andrea; Bassetti, D.
abstract
Drug-associated dysfunction of mitochondria (mt) is believed to play a role in the aetiology of the various adverse symptoms that occur in HIV-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Switching to drugs with less mt toxicity may be of benefit in HIV-infected patients receiving antiretroviral therapy containing stavudine (d4T), but the efficacy and the safety of this strategy in paediatric population is unknown. We have analysed the effect of the switch from d4T to tenofovir (TDF), paying particular attention to the maintenance of the immunovirological status and lipid profile.
2005
- Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin
[Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Roat, Erika; E., Lugli; Nemes, Elisa; Nasi, Milena; Pinti, Marcello; M. I. G., Fernandez; E. L., Cooper; Cossarizza, Andrea
abstract
We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H2O2), superoxide anion (O-2), reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H2O2 levels), whereas for long periods it showed a pro-oxidant activity (increase in O-2). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.
2005
- Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naive HIV-positive patients.
[Articolo su rivista]
Nasi, Milena; Pinti, Marcello; Bugarini, R; Troiano, Leonarda; Lugli, E; Bellodi, C; Mussini, Cristina; Borghi, V; Trenti, T; Balli, Fiorella; Esposito, Roberto; Cossarizza, Andrea
abstract
Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naive HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using X-2 test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p=0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.
2005
- Impairment of recent thymic emigrants in HCV infection.
[Articolo su rivista]
Cianci, R; Nasi, Milena; Pinti, Marcello; Starnino, S; Florio, A; Cammarota, G; De Luca, A; Cauda, R; Grieco, A; Rapaccini, G; Gasbarrini, G; Cossarizza, Andrea; Pandolfi, F.
abstract
Hepatitis C Virus (HCV) often has a more favorable course in younger patients. Considering the involution of the thymic function with age, we investigated the output of recent thymic emigrants (RTE) in HCV patients. To evaluate RTE, we used a competitive quantitative PCR in order to determine the percentages of cells with cj-T cell receptor excision circles (TREC). This study was performed in 14 HCV patients at diagnosis and before any anti-HCV treatment. The results obtained in this group were compared to those obtained in a group of age-matched controls. We found that in the 14 HCV patients naive for anti-HCV treatment the mean percentage of cj-TREC was 3%. We could not detect a correlation between the percentages of cj-TREC and age or patients' viremia. In contrast, in the 26 age-matched controls mean percentage of cj-TREC was 5.6% (P=0.01). Our study describes a novel immune defect in HCV patients. Additional studies are needed to get further insight in the possible role of TREC defect in the pathogenesis and prognosis of the disease.
2005
- MMP-7 promoter polymorphisms do not influence CD4+ recovery and changes in plasma viral load during antiretroviral therapy for HIV-1 infection.
[Articolo su rivista]
E., Lugli; Pinti, Marcello; Nasi, Milena; Troiano, Leonarda; Prada, Nicole; Mussini, Cristina; V., Borghi; Esposito, Roberto; Cossarizza, Andrea
abstract
Matrix metalloproteinase-7 (MMP-7) generates soluble Fas Ligand (FasL), which is involved in the apoptotic loss of CD4(+) T cells during HIV infection. We evaluated whether two polymorphisms in MMP-7 promoter could influence CD4(+) recover in response to antiretroviral therapy, and found that these polymorphisms are ineffective.
2004
- Balanced regulation of mRNA production for Fas and Fas ligand in lymphocytes from centenarians: how the immune system starts its second century.
[Articolo su rivista]
Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; Ferraresi, Roberta; Mussi, Chiara; Bellodi, C; Salvioli, Gianfranco; Cossarizza, Andrea
abstract
The functionality of the immune system during aging is crucial for protection against the most common age-related diseases. Apoptosis plays a central role in the senescence of the immune system, as evidenced by the increased plasma membrane expression of a key molecule like Fas protein. We analyzed the mRNA levels of different forms of Fas (total [tFas] and membrane [mFas]) and of its ligand (FasL) in peripheral blood lymphocytes from centenarians, the best example of successful aging, who were compared with young and middle-aged donors.
2004
- Complementary and alternative medicine during HIV infection.
[Relazione in Atti di Convegno]
Nasi, Milena; Pinti, Marcello; Troiano, Leonarda; Cossarizza, Andrea
abstract
According to the Joint United Nations Program of HIV/AIDS (UNAIDS) and the World Health Organization (WHO) (Joint United Nations Program of HIV/AIDS, 2001), as of the end of 2001, there were about 40 million adults and children living with human immunod-eficiency virus (HIV) infection. This total does not include the 20 million people around the world who already died of AIDS. Of the 40 million currently alive, 37.2 are adults, 17.6 are women, and more than 2.7 are children. In 2001, there were 5 million new cases of HIV infection in the world, and 3 million AIDS related deaths. The large majority (almost three quarters) live in Sub-Saharan Africa where the prevalence rate of the infection among adults is 8.4%; more than 55% of infected individuals are women. The second major pocket of HIV infection is in South and Southeast Asia, with more than 6 million people infected. In North America where the epidemic was first described, there are 940,000 individuals who are HIV- , and in Western Europe, 540,000. Furthermore, South America, China, and East-ern Europe are characterized by a rapid increase in infection rates. These dramatic numbers clearly indicate that the fight against HIV/AIDS is an absolute health, social, economical and political priority in all parts of the world.
2004
- Mitochondria analysis for investigating toxicity of antiretroviral drugs.
[Abstract in Atti di Convegno]
Troiano, Leonarda; Ferraresi, Roberta; Nemes, Elisa; Lugli, E; Rossi, D; Gualdi, E; Nasi, Milena; Galluzzi, Lorenzo; Prada, Nicole; Maffei, Stefania; Pinti, Marcello; Cossarizza, Andrea
abstract
Mitochondria are the key organelles in intracellular energy production, but performseveral other biologic functions and carry factors involved in cell apoptosis.Both HIV infection and antiretroviral nucleoside analogues (NRTI) can affectmitochondrial (mt) function and DNA content. Several side effects occurring inindividuals with HIV infection who are on antiretroviral therapy have been linkedto mitochondrial injury and dysfunction. Clinical studies of NRTIs, while collectingadverse event data, have not specifically evaluated mitochondrial toxicityin a systematic way. In vitro studies have demonstrated that NRTIs may differ intheir effects on mitochondria activities and may affect mtDNA content in differentcell lines in different ways, and several models are actually employed to betterunderstand not only the mechanism(s) of action of antiretroviral compounds,but also their possible side effects. Accordingly, it has been hypothesized thatclinical syndromes associated with toxicity to these agents are likely caused, atleast in part, by drug-related mitochondria alterations. Dideoxy-NRTIs have thegreatest affinity for mtDNA polymerase-γ, the enzyme responsible for mtDNAreplication, whereas other nucleoside analogues may influence mitochondrialfunction also through other mechanisms. By using cytometric assays to analyzemt or cell functionality (mt membrane potential by JC-1, mt mass by Mitotrackergreen, cell viability and apoptosis by classical stainings) along with originalmolecular biology techniques based upon real time PCR for determining mtDNAcontent, we have investigated the effects of different drugs commonly employedin anti-HIV therapy. We have used several human cell lines of different origin(lymphocytic such as CEM; monocytic, U937 or hepatocytic, HepG2). We foundthat the aforementioned parameters can be altered by antiretroviral drugs, with adifferent extent in the cell lines we studied. In general, stavudine seemed morepotent than zidovudine or didanosine in inducing damages to mt function ormtDNA. Antiretroviral drugs of the protease inhibitor category (such as indinavir)had no effects on any mt parameter we analyzed. From our experience, it can beconcluded that the choice of adequate molecular or cellular techniques and modelsis important in evaluating differences in drug activity that are related to theimpact of antiretroviral agents on mitochondria, as well as in understanding theside effects of such drugs on different cells, organs or systems.
2003
- Apoptosis-resistant phenotype in HL-60-derived cells HCW-2 is related to changes in expression of stress-induced proteins that impact on redox status and mitochondrial metabolism
[Articolo su rivista]
S., Salvioli; G., Storci; Pinti, Marcello; Quaglino, Daniela; L., Moretti; M., Merlo Pich; G., Lenaz; S., Filosa; A., Fico; M., Bonafe; D., Monti; Troiano, Leonarda; Nasi, Milena; Cossarizza, Andrea; C., Franceschi
abstract
The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapic drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.
2003
- Development of real time PCR assays for the quantification of Fas and FasL mRNA levels in lymphocytes: studies on centenarians
[Articolo su rivista]
Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; Monterastelli, Elena; L., Moretti; C., Bellodi; A., Mazzacani; Mussi, Chiara; Salvioli, Gianfranco; Cossarizza, Andrea
abstract
Apoptosis plays a central role in the homeostasis of the immune system. During aging, there is an altered regulation of pivotal molecules that are responsible for the regulation of this type of cell death, such as those of the Fas/FasL system. Understanding the regulation of these genes can help to clarify, at least in part, the age-related changes that occur in immune cells. We have developed an original real time PCR assay for quantification of mRNA for Fas and FasL, and have studied a group of young donors, middle aged subjects and centenarians. We have found that the production of Fas mRNA is greatly increased in resting lymphocytes from centenarians; such an increase follows an age-related trend. On the contrary, the production of mRNA for the molecule, which is the natural ligand of Fas, i.e. FasL, is consistently reduced. Our preliminary results suggest that during aging a subtle balance in the production of molecules that cause apoptosis could exist, and that, in order to avoid an excessive death of immune cells, a still unknown mechanism could compensate the increase of Fas with the reduction of FasL. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.
2003
- Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1
[Articolo su rivista]
Pinti, Marcello; Biswas, P; Troiano, L; Nasi, Milena; Ferraresi, Roberta; Mussini, Cristina; Vecchiet, J; Esposito, Roberto; Paganelli, R; Cossarizza, Andrea
abstract
Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-alpha plus cycloheximide, and gamma-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules. Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.
2003
- Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which correlation with the patient?
[Articolo su rivista]
Pinti, Marcello; L., Troiano; Nasi, Milena; R., Ferraresi; J., Dobrucki; Cossarizza, Andrea
abstract
Currently, drugs have been synthesised that can significantly delay the course of several viral infections, including those provoked by HBV, HCV or HIV, but that display consistent side effects, including toxicity for organelles such as mitochondria. Several in vitro models and techniques have been developed to analyse the effects of such compounds. HepG2 cells (from human hepatoma) are an excellent model to investigate mitochondrial (mt) toxicity because of their high content of organelles and mtDNA, and actually different investigators are indeed using such cells. Studies in vitro on cell lines are relatively easy, but it is necessary to be careful. in the interpretation of data, which are usually obtained on continuously growing, tumour cells, quite different from normal, resting, non-neoplastic cells collected from a patient. Direct analysis of drug-induced mt damage in patients is extremely more complex than that performed using in vitro models because of the difficulty to obtain adequate cells or to have discrete amounts of biological material, the status of the patient at the moment of cell collection, the use of an adequate assay and its correct execution, and finally the possibility to find sex- and age-matched healthy controls as source of reference parameters.
2003
- MDR1 C3435T genetic polymorphism does not influence the response to antiretroviral therapy in drug-naive HIV-positive patients
[Articolo su rivista]
Nasi, Milena; V., Borghi; Pinti, Marcello; C., Bellodi; E., Lugli; S., Maffei; L., Troiano; Richeldi, Luca; Mussini, Cristina; Esposito, Roberto; Cossarizza, Andrea
abstract
P-glycoprotein, a membrane-localized protein transporter, codified by the MDR1 gene, influences the response to pharmacological treatments, including antiretroviral drugs. MDR1 polymorphism C3435T is correlated with the functionality of the protein. We investigated the influence of this polymorphism in the reconstitution of the peripheral C134 T cell pool in 149 drug-naive HIV-positive patients starting highly active antiretroviral therapy. The MDR1 C3435T polymorphism did not influence response to therapy, suggesting no disadvantages for individuals with a different genotype.
2002
- Early changes in intramitochondrial cardiolipin distribution during apoptosis.
[Articolo su rivista]
Garcia Fernandez, M.; Troiano, L.; Moretti, L.; Nasi, Milena; Pinti, Marcello; Salvioli, S.; Dobrucki, J.; Cossarizza, Andrea
abstract
Cardiolipin (CL) is essential for the functionality of several mitochondrial proteins. Its distribution between the inner and outer leaflet of the mitochondrial internal membrane is crucial for ATP synthesis. We have investigated alterations in CL distribution during the early phases of apoptosis. Using two classical models (staurosporine-treated HL-60 cells and tumor necrosis factor alpha-treated U937 cells), we found that in apoptotic cells CL moves to the outer leaflet of mitochondrial inner membrane in a time-dependent manner. This occurs before the appearance of apoptosis markers such as plasma-membrane exposure of phosphatidylserine, changes in mitochondrial membrane potential, DNA fragmentation, but after the production of reactive oxygen species. The exposure of a phospholipid on the outer surface during apoptosis thus occurs not only at the plasma membrane level but also in mitochondria, reinforcing the hypothesis of mitoptosis as a crucial regulating system for programmed cell death, also occurring in cancer cells after treatment with antineoplastic agents.
2002
- Features of 'CD4-exploders', HIV-positive patients with an optimal immune reconstitution after potent antiretroviral therapy
[Articolo su rivista]
Mussini, Cristina; Pinti, Marcello; Borghi, V; Nasi, Milena; Amorico, G; Monterastelli, E; Moretti, L; Troiano, Leonarda; Esposito, Roberto; Cossarizza, Andrea
abstract
Objective: To identify crucial immunological characteristics of a group of patients, defined ´CD4-exploders´, who were able to fully reconstitute their immune system after receiving highly active antiretroviral therapy (HAART). Patients: Among a population of 540 HIV-positive patients treated with HAART, six individuals were identified who experienced a nadir of less than 85 X 10(6) CD4+ cells/l, had major opportunistic infections (four out of six), started HAART in 1996 or 1997, and showed a rapid and relevant CD4+ lymphocyte increase (mainly due to virgin cells), in some cases regardless of virological control. Methods: Enzyme-linked immunosorbent assay for the determination of interleukin (IL)-7 plasma levels; flow cytometry to analyse surface antigens and CD127 (IL-7 receptor alpha-chain) expression; quantitative-competitive (QC) PCR for detecting cells containing T-cell receptor rearrangement excision circles (TREC) chest-computed tomography (CT) to analyse thymus volume and content. Results: In ´CD4-exploders´, high levels of TREC+ lymphocytes were found among CD4+ T cells, which also contained a high percentage of naive cells. However, CT revealed a dramatic depletion of intrathymic lymphoid tissue. Plasma levels of IL-7 were significantly high. Most CD4+ and CD8+ T lymphocytes expressed CD127, whose level was similar to that of healthy donors. CD127 expression on CD8+ lymphocytes was markedly higher than in HIV-positive individuals treated with the same therapy or in patients with AIDS. Conclusion: In ´CD4-exploders´, HAART-incluced reconstitution of the T-cell compartment is independent from thymus volume, and is favoured by the upregulation of the IL-7/IL-7 receptor system.
2002
- Genetic polymorphisms of Fas (CD95) and FasL (CD178) in human longevity: studies on centenarians
[Articolo su rivista]
Pinti, Marcello; Troiano, Leonarda; Nasi, Milena; L., Moretti; E., Monterastelli; A., Mazzacani; Mussi, Chiara; Ventura, Paolo; F., Olivieri; C., Franceschi; Salvioli, Gianfranco; Cossarizza, Andrea
abstract
Apoptosis plays a crucial role in immunosenescence, as also evidenced by the increased expression of Fas in lymphocytes from aged people. However, little is known about the genetic regulation of Fas and its ligand, FasL. We have studied their polymorphisms in 50 centenarians and 86 young donors living in Northern Italy. The first Fas polymorphism; at position -670, has in Caucasian a heterozigosity of 51%; the second, at -1377 position, has the wild type allele (G) with a very high frequency (83%) respect to the mutant allele. Genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found as far as two FasL polymorphisms (IVS2nt-124 and IVS3nt169) are concerned. On the whole, our data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity.
2002
- The evolution of cell killing: when a target cell became “invited” to choose how to die.
[Relazione in Atti di Convegno]
Nasi, Milena; Pinti, Marcello; Troiano, Leonarda; Moretti, L.; Cooper, E. L.; Cossarizza, Andrea
abstract
During evolution, in the world of cytotoxic reactions a new mechanism has emerged that was based upon the direct "responsibility" of targets in their death. In other words, effector cells thought targets to use their own genetic material to trigger their suicide. To investigate this hypothesis, we have used an experimental model represented by Eisenia foetida coelomocytes that kill human cells, and asked whether coelomocytes can trigger apoptosis, or are able to provoke their death via necrosis, or even both, Using a strategy based upon a technique we have developed, that evaluates the expression of different forms of Fas (CD95/APO-1, i.e. the proapoptotic membrane form and the anti-apoptotic soluble form) mRNA in target cells, we tested the capacity of either coelomocytes from E. foetida or supernatant from E. foetida coelomocyte cultures to kill human cell lines of different origin. When target cells were incubated with coelomocytes, an upregulation of the membrane form of Fas occurred, along with an increase of the total form. On the contrary, cells treated with supernatant (containing cytotoxic molecules) had a significant reduction of the production of both forms of Fas mRNA, suggesting that mechanisms devoted to triggering of apoptosis were downregulated. Our data suggest that the production of soluble mediators, that could be considered the ancestors of humoral immunity, causes death of the foreign cell because of an aspecific activity of lytic molecules, that likely bind target membrane for physicochemical reasons, and determine necrosis. The development of cellular mechanisms to kill targets, i.e. likely representing the onset of cellular immunity, could act by modulating the expression of genes involved in apoptosis, determining an increase of total Fas expression.
2001
- Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age.
[Articolo su rivista]
Viganò, A; Pinti, Marcello; Nasi, Milena; Moretti, L; Balli, Fiorella; Mussini, Cristina; Bricalli, D; Sala, N; Bugarini, R; Vella, S; Principi, N; Cossarizza, Andrea
abstract
BACKGROUND:
Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear.
OBJECTIVES:
We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection.
METHODS:
Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential.
RESULTS:
Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage.
CONCLUSION:
Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.
2000
- Quantitation of CD95 and CD95L mRNA expression in chronic and acute HIV-1 infection by competitive RT-PCR
[Articolo su rivista]
Pinti, Marcello; Nasi, Milena; Moretti, L; Mussini, Cristina; Petrusca, D; Esposito, Roberto; Cossarizza, Andrea
abstract
Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by increased immune cell apoptosis. Apoptosis can be triggered by signals that arise from within the cell, or by signals that are elicited by binding of extracellular death ligands to their death receptors, most of which belong to the tumor necrosis factor (TNF)-receptor family, such as CD95 (Fas/Apo-1). In immune cells the oligomerization of CD95, induced by its ligand CD95L, and the recruitment of different intracytoplasmic molecules that in turn activate FLICE/caspase 8 are crucial. To study the role of CD95/CD95L interactions during HIV-1 infection, we developed an original method based upon quantitative-competitive (QC) RT-PCR that allowed us to quantify the amounts of mRNA coding for the total (tCD95) and membrane (mCD95) forms of CD95. We first studied the expression of different forms of CD95 mRNA in a classical model of chronic HIV infection using two infected cell lines of different origin-lymphocytic (ACH-2) or monocytic (U1). We have shown that infected cells of monocytic origin preferentially produce the protective (soluble) form of CD95, and no detectable CD95L mRNA, while lymphoid cells produce more mRNA for the membrane form of CD95 (which triggers apoptosis) along with low but detectable amounts of CD95L mRNA. One can hypothesize that a complex balance exists between pro-apoptotic events, perhaps triggered by the host to limit viral production, and anti-apoptotic events likely triggered by the virus to increase its production and survival. In cells of monocytic origin, which act as a reservoir for the virus, the anti-apoptotic molecules are favored; in cells of lymphocytic origin, molecules with an apoptotic meaning are prevalent.