Publications by authors named "Maurizio Fraziano"

33 Publications

Editorial: Exploiting Novel Combined Host- and Pathogen-Directed Therapies for Combating Bacterial Multidrug Resistance.

Front Immunol 2020 4;11:616486. Epub 2020 Nov 4.

Department of Biology, University of Rome Tor Vergata, Rome, Italy.

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http://dx.doi.org/10.3389/fimmu.2020.616486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672020PMC
November 2020

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress.

EMBO Rep 2021 Jan 27;22(1):e50500. Epub 2020 Nov 27.

Department of Biology, Tor Vergata University, Rome, Italy.

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.
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http://dx.doi.org/10.15252/embr.202050500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788447PMC
January 2021

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response.

Front Immunol 2020 2;11:532225. Epub 2020 Oct 2.

Dipartimento di Biologia, Università degli Studi di Roma "Tor Vergata", Roma, Italy.

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in and models of MDR infection. Our results show that the treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR significantly reduces NF-κB activation and the production of TNF-α, IL-1β, and IL-6, while increasing IL-10 and TGF-β levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR . The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF.
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http://dx.doi.org/10.3389/fimmu.2020.532225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562816PMC
October 2020

Characterization of vB_StuS_MMDA13, a Newly Discovered Bacteriophage Infecting the Agar-Degrading Species .

Viruses 2020 08 15;12(8). Epub 2020 Aug 15.

Department of Biology, Tor Vergata University, 00133 Rome, Italy.

Members of genus have gained a notable interest for their use in a wide range of biotechnological applications, ranging from bioremediation to the production of valuable compounds of industrial interest. To date, knowledge on phages targeting spp. are still scarce. Here, we describe and characterize a lytic bacteriophage, named vB_StuS_MMDA13, able to infect the MCT13 type strain. Physiological characterization demonstrated that vB_StuS_MMDA13 has a narrow host range, a long latency period, a low burst size, and it is overall stable to both temperature and pH variations. The phage has a double-stranded DNA genome of 63,743 bp, with 89 open reading frames arranged in two opposite arms separated by a 1186 bp non-coding region and shows a very low global similarity to any other known phages. Interestingly, vB_StuS_MMDA13 is endowed with an original nucleotide modification biosynthetic gene cluster, which greatly differs from those of its most closely related phages of the genus. vB_StuS_MMDA13 is the first characterized lytic bacteriophage of the family infecting members of the genus.
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http://dx.doi.org/10.3390/v12080894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472734PMC
August 2020

The Urgent Need for Novel Antimicrobial Agents and Strategies to Fight Antibiotic Resistance.

Antibiotics (Basel) 2019 Dec 6;8(4). Epub 2019 Dec 6.

Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy.

Antibiotic resistance in bacterial pathogens has currently reached very high and alarming levels [...].
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http://dx.doi.org/10.3390/antibiotics8040254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963704PMC
December 2019

Adipocyte metabolism is improved by TNF receptor-targeting small RNAs identified from dried nuts.

Commun Biol 2019 21;2:317. Epub 2019 Aug 21.

1Department of Biology, University of Rome Tor Vergata, Rome, Italy.

There is a growing interest in therapeutically targeting the inflammatory response that underlies age-related chronic diseases including obesity and type 2 diabetes. Through integrative small RNA sequencing, we show the presence of conserved plant miR159a and miR156c in dried nuts having high complementarity with the mammalian TNF receptor superfamily member 1a (Tnfrsf1a) transcript. We detected both miR159a and miR156c in exosome-like nut nanovesicles (NVs) and demonstrated that such NVs reduce Tnfrsf1a protein and dampen TNF-α signaling pathway in adipocytes. Synthetic single-stranded microRNAs (ss-miRs) modified with 2'--methyl group function as miR mimics. In plants, this modification naturally occurs on nearly all small RNAs. 2'--methylated ss-miR mimics for miR156c and miR159a decreased Tnfrsf1a protein and inflammatory markers in hypertrophic as well as TNF-α-treated adipocytes and macrophages. miR156c and miR159a mimics effectively suppress inflammation in mice, highlighting a potential role of plant miR-based, single-stranded oligonucleotides in treating inflammatory-associated metabolic diseases.
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http://dx.doi.org/10.1038/s42003-019-0563-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704100PMC
April 2020

Immunization With Antigens Encapsulated in Phosphatidylserine Liposomes Improves Protection Afforded by BCG.

Front Immunol 2019 12;10:1349. Epub 2019 Jun 12.

Institute for Infection and Immunity, St. George's University of London, London, United Kingdom.

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol , we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.
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http://dx.doi.org/10.3389/fimmu.2019.01349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598733PMC
October 2020

First Case of Patient With Two Homozygous Mutations in and Genes Presenting With Pyogenic Bacterial Infections, Elevated IgE, and Persistent EBV Viremia.

Front Immunol 2019 14;10:130. Epub 2019 Feb 14.

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù, Rome, Italy.

We described for the first time a female patient with the simultaneous presence of two homozygous mutations in and genes presenting with pyogenic bacterial infections, elevated IgE, and persistent EBV viremia. In addition to defective TLR/IL1R-signaling, we described novel functional alterations into the myeloid compartment. In particular, we demonstrated a defective production of reactive oxygen species exclusively in monocytes upon stimulation, the inability of immature mono-derived DCs (iDCs) to differentiate into mature DCs (mDCs) and the incapacity of mono-derived macrophages (MDMs) to resolve BCG infection . Our data do not provide any evidence for digenic inheritance in our patient, but rather for the association of two monogenic disorders. This case illustrates the importance of using next generation sequencing (NGS) to determine the most accurate and early diagnosis in atypical clinical and immunological phenotypes, and with particular concern in consanguineous families. Indeed, besides the increased susceptibility to recurrent invasive pyogenic bacterial infections due to MYD88 deficiency, the identification of mutations underline the risk of developing invasive fungal infections emphasizing the careful monitoring for the occurrence of fungal infection and the opportunity of long-term antifungal prophylaxis.
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http://dx.doi.org/10.3389/fimmu.2019.00130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383679PMC
December 2019

Hydroalcoholic extract from Origanum vulgare induces a combined anti-mycobacterial and anti-inflammatory response in innate immune cells.

PLoS One 2019 4;14(3):e0213150. Epub 2019 Mar 4.

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

In nature, many plants or their extracted compounds have been found to possess anti-inflammatory features and therapeutic properties against infectious as well as non-infectious diseases, including cancer. In this study, we analysed the immunomodulatory effects on innate immune cells of hydroalcoholic extract from Origanum vulgare L. ssp. hirtum (HyE-Ov), a plant traditionally known for its anti-oxidative properties. The effects of HyE-Ov were tested on human monocyte derived dendritic cells (DC), type-1 (M1) and type-2 macrophages (M2) infected with M. bovis Bacille Calmette-Guérin (BCG), used as a model of persistent intracellular bacterium. DC, M1 and M2 treated with HyE-Ov significantly enhanced their mycobactericidal activity, which was associated with phagosomal acidification in M1 and M2 and increase of phagosomal, but not mitochondrial ROS production in M1, M2, and DC. Treatment of BCG-infected DC with HyE-Ov significantly reduced TNF-α and IL-12 production and increased TGF-β synthesis. Finally, experiments were repeated using eight different HPLC fractions of HyE-Ov. Results showed that the capability to activate anti-microbial and anti-inflammatory response is shared by different fractions, suggesting that diverse bioactive molecules are present within the hydroalcoholic extract. Altogether, these results show that HyE-Ov promotes anti-mycobacterial innate immunity and limits inflammatory response in vitro and suggest that this plant extract may be exploitable as phytocomplex or nutraceutical for novel host-directed therapeutic approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213150PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398838PMC
December 2019

The RNA binding protein Sam68 controls T helper 1 differentiation and anti-mycobacterial response through modulation of miR-29.

Cell Death Differ 2019 06 26;26(6):1169-1180. Epub 2018 Sep 26.

Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy.

Polarization of naive T cells into interferon (IFN)-γ-producing T helper 1 (Th1) cells is an essential event in the inflammatory response to pathogens. Herein, we identify the RNA binding protein Sam68 as a specific modulator of Th1 differentiation. Sam68-knockout (ko) naive T cells are strongly defective in IL-12-mediated Th1 polarization and express low levels of T-bet and Eomes. Consequently, Sam68-ko Th1 cells are significantly impaired in IFN-γ production. Moreover, we found that Sam68 is required for the induction of an inflammatory Th1 response during Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, thus limiting bacterial dissemination in the lungs. Mechanistically, Sam68 directly binds to the microRNA miR-29, a negative regulator of Th1 response, and inhibits its expression during BCG infection. These findings uncover a novel post-transcriptional mechanism required for the Th1-mediated defense against intracellular pathogens and identify a new function for Sam68 in the regulation of the immune response.
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http://dx.doi.org/10.1038/s41418-018-0201-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748099PMC
June 2019

Characterization of vB_Kpn_F48, a Newly Discovered Lytic Bacteriophage for Klebsiella pneumoniae of Sequence Type 101.

Viruses 2018 09 9;10(9). Epub 2018 Sep 9.

Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.

Resistance to carbapenems in , including , represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for isolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of , order , on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the subfamily, which we tentatively named "". Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications.
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http://dx.doi.org/10.3390/v10090482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163469PMC
September 2018

The Multirole of Liposomes in Therapy and Prevention of Infectious Diseases.

Front Immunol 2018 5;9:155. Epub 2018 Feb 5.

Dipartimento di Biologia, Università degli Studi di Roma "Tor Vergata", Rome, Italy.

Liposomes are closed bilayer structures spontaneously formed by hydrated phospholipids that are widely used as efficient delivery systems for drugs or antigens, due to their capability to encapsulate bioactive hydrophilic, amphipathic, and lipophilic molecules into inner water phase or within lipid leaflets. The efficacy of liposomes as drug or antigen carriers has been improved in the last years to ameliorate pharmacokinetics and capacity to release their cargo in selected target organs or cells. Moreover, different formulations and variations in liposome composition have been often proposed to include immunostimulatory molecules, ligands for specific receptors, or stimuli responsive compounds. Intriguingly, independent research has unveiled the capacity of several phospholipids to play critical roles as intracellular messengers in modulating both innate and adaptive immune responses through various mechanisms, including (i) activation of different antimicrobial enzymatic pathways, (ii) driving the fusion-fission events between endosomes with direct consequences to phagosome maturation and/or to antigen presentation pathway, and (iii) modulation of the inflammatory response. These features can be exploited by including selected bioactive phospholipids in the bilayer scaffold of liposomes. This would represent an important step forward since drug or antigen carrying liposomes could be engineered to simultaneously activate different signal transduction pathways and target specific cells or tissues to induce antigen-specific T and/or B cell response. This lipid-based host-directed strategy can provide a focused antimicrobial innate and adaptive immune response against specific pathogens and offer a novel prophylactic or therapeutic option against chronic, recurrent, or drug-resistant infections.
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http://dx.doi.org/10.3389/fimmu.2018.00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807682PMC
April 2019

A method permissive to fixation and permeabilization for the multiparametric analysis of apoptotic and necrotic cell phenotype by flow cytometry.

Cytometry A 2017 11 26;91(11):1115-1124. Epub 2017 Oct 26.

Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Roma, Italy.

Annexin-V/propidium iodide method (A-V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. However, A-V/PI does not permit fixation and/or permeabilization of cells making impossible evaluation of intracellular markers, restricting the analysis in a narrow time frame after staining and excluding the possibility to study pathogen-infected cells. We developed a method permitting fixation and permeabilization of stained cells: Fixed Apoptotic/Necrotic (FAN) cells test. FAN relies on the same principle of A-V/PI, but uses reagents that maintain their binding and fluorescence characteristics after fixation/permeabilization: a fluorochrome-labeled anti-phosphatidylserine antibody and fluorescent amine-binding dyes. FAN was tested to discriminate apoptotic and necrotic cells using different stimuli on several cell types and results were always comparable to those obtained using A-V/PI. FAN, unlike A-V/PI, permitted to correlate cell death with intracellular and surface markers expression and to perform cytometry even two weeks after sample preparation. As fixation of stained cells inactivates infective pathogens, we used FAN in an in vitro model of Mycobacterium tuberculosis (Mtb) infection of macrophages to monitor cellular infection and cell death induction. Using a red-fluorescent Mtb, fluorochrome labeled anti-TNF-α and anti-MHC class II monoclonal antibodies, FAN permitted to establish that the extent of macrophage death correlates with intracellular Mtb content and that dying cells accumulate TNF-α and down-modulate MHC class II molecules. Results suggest that FAN may represent an additional tool to study programmed cell death particularly useful when fixation procedures are required for a safe infected sample analysis or to comparatively analyze multiple samples. © 2017 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23268DOI Listing
November 2017

Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance.

Sci Rep 2017 03 27;7:45120. Epub 2017 Mar 27.

Department of Biology, University of Rome Tor Vergata Rome, Italy.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.
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http://dx.doi.org/10.1038/srep45120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366871PMC
March 2017

Phosphodiesterase Type 5 Inhibitor Sildenafil Decreases the Proinflammatory Chemokine CXCL10 in Human Cardiomyocytes and in Subjects with Diabetic Cardiomyopathy.

Inflammation 2016 Jun;39(3):1238-52

Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.

T helper 1 (Th1) type cytokines and chemokines are bioactive mediators in inflammation underling several diseases and co-morbid conditions, such as cardiovascular and metabolic disorders. Th1 chemokine CXCL10 participates in heart damage initiation/progression; cardioprotection has been recently associated with sildenafil, a type 5 phosphodiesterase inhibitor. We aimed to evaluate the effect of sildenafil on CXCL10 in inflammatory conditions associated with diabetic cardiomyopathy. We analyzed: CXCL10 gene and protein in human cardiac, endothelial, and immune cells challenged by pro-inflammatory stimuli with and without sildenafil; serum CXCL10 in diabetic subjects at cardiomyopathy onset, before and after 3 months of treatment with sildenafil vs. placebo. Sildenafil significantly decreased CXCL10 protein secretion (IC50 = 2.6 × 10(-7)) and gene expression in human cardiomyocytes and significantly decreased circulating CXCL10 in subjects with chemokine basal level ≥ 930 pg/ml, the cut-off value as assessed by ROC analysis. In conclusion, sildenafil could be a pharmacologic tool to control CXCL10-associated inflammation in diabetic cardiomyopathy.
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http://dx.doi.org/10.1007/s10753-016-0359-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883282PMC
June 2016

The case of an APDS patient: Defects in maturation and function and decreased in vitro anti-mycobacterial activity in the myeloid compartment.

Clin Immunol 2017 05 28;178:20-28. Epub 2015 Dec 28.

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù, Rome, Italy. Electronic address:

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patient's B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients.
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http://dx.doi.org/10.1016/j.clim.2015.12.008DOI Listing
May 2017

Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis.

PLoS One 2015 29;10(5):e0127279. Epub 2015 May 29.

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127279PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449037PMC
April 2016

In vitro analysis of pyrogenicity and cytotoxicity profiles of flex sensors to be used to sense human joint postures.

Sensors (Basel) 2014 Jul 1;14(7):11672-81. Epub 2014 Jul 1.

Department of Biology, University of Tor Vergata, Via della Ricerca Scientifica, 1-00173 Rome, Italy.

Flex sensors can be usefully adopted as mechanical-electrical transducers to measure human joint movements, since their electrical resistance varies proportionally to the angle assumed by the joint under measure. Over time, these sensors have been investigated in terms of mechanical and electrical behavior, but no reports have detailed the possibility of their adoption not just on top but under the human skin of the joint. To this aim, our work investigated in vitro the pyrogenic potential and cytotoxicity of some commercially available flex sensors as a first step toward the necessary requirements regarding their biocompatibility, to predict possible foreign body reactions when used in vivo. Results demonstrated that some specific flex sensors satisfy such requirements.
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http://dx.doi.org/10.3390/s140711672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168521PMC
July 2014

Dormant Mycobacterium tuberculosis fails to block phagosome maturation and shows unexpected capacity to stimulate specific human T lymphocytes.

J Immunol 2013 Jul 3;191(1):274-82. Epub 2013 Jun 3.

Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, 00161 Rome, Italy.

Dormancy is defined as a stable but reversible nonreplicating state of Mycobacterium tuberculosis. It is currently thought that dormant M. tuberculosis (D-Mtb) is responsible for latent tuberculosis (TB) infection. Recently, D-Mtb was also shown in sputa of patients with active TB, but the capacity of D-Mtb to stimulate specific immune responses was not investigated. We observed that purified protein derivative-specific human CD4(+) T lymphocytes recognize mycobacterial Ags more efficiently when macrophages are infected with D-Mtb instead of replicating M. tuberculosis (R-Mtb). The different Ag recognition occurs even when the two forms of mycobacteria equally infect and stimulate macrophages, which secrete the same cytokine pattern and express MHC class I and II molecules at the same levels. However, D-Mtb but not R-Mtb colocalizes with mature phagolysosome marker LAMP-1 and with vacuolar proton ATPase in macrophages. D-Mtb, unlike R-Mtb, is unable to interfere with phagosome pH and does not inhibit the proteolytic efficiency of macrophages. We show that D-Mtb downmodulates the gene Rv3875 encoding for ESAT-6, which is required by R-Mtb to block phagosome maturation together with Rv3310 gene product SapM, previously shown to be downregulated in D-Mtb. Thus, our results indicate that D-Mtb cannot escape MHC class II Ag-processing pathway because it lacks the expression of genes required to block the phagosome maturation. Data suggest that switching to dormancy not only represents a mechanism of survival in latent TB infection, but also a M. tuberculosis strategy to modulate the immune response in different stages of TB.
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http://dx.doi.org/10.4049/jimmunol.1202900DOI Listing
July 2013

Mycobacterium tuberculosis may escape helper T cell recognition by infecting human fibroblasts.

Hum Immunol 2013 Jun 28;74(6):722-9. Epub 2013 Feb 28.

Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Roma, Italy.

The host immune response can limit Mycobacterium tuberculosis (Mtb) spreading in primary tuberculosis (TB) without eradicating all bacilli, which can persist causing latent TB infection and are responsible for reactivation TB. Persistent Mtb is confined to granulomas within phagocytes, but it is also found in other non-immune cells. We focused on fibroblasts since these cells participate to the granuloma formation and were shown to be infected in latent TB infections. We show that in vitro both Mtb and Bacille Calmette-Guérin actively replicate in human fibroblasts. Mycobacterial infection of fibroblasts causes a significant inhibition of interferon (IFN)-γ induced membrane expression of major histocompatibility complex class II molecules in these cells. The functional consequence of in vitro infection is a significant reduction of the fibroblast capacity to present peptides and soluble proteins to autologous specific CD4(+) T cell clones. Moreover, fibroblasts are capable of presenting antigen derived from the processing of heat-killed Mtb, but not from viable Mtb. Data indicate that IFN-γ treated fibroblasts are capable of presenting antigens derived from the processing of whole bacteria in addition to the capacity to present peptides and isolated proteins. Interestingly, Mtb infected fibroblasts lose this capacity, suggesting that Mtb may evade T helper immune surveillance by infecting fibroblasts.
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http://dx.doi.org/10.1016/j.humimm.2013.02.005DOI Listing
June 2013

B-Pred, a structure based B-cell epitopes prediction server.

Adv Appl Bioinform Chem 2012 25;5:11-21. Epub 2012 Jul 25.

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

The ability to predict immunogenic regions in selected proteins by in-silico methods has broad implications, such as allowing a quick selection of potential reagents to be used as diagnostics, vaccines, immunotherapeutics, or research tools in several branches of biological and biotechnological research. However, the prediction of antibody target sites in proteins using computational methodologies has proven to be a highly challenging task, which is likely due to the somewhat elusive nature of B-cell epitopes. This paper proposes a web-based platform for scoring potential immunological reagents based on the structures or 3D models of the proteins of interest. The method scores a protein's peptides set, which is derived from a sliding window, based on the average solvent exposure, with a filter on the average local model quality for each peptide. The platform was validated on a custom-assembled database of 1336 experimentally determined epitopes from 106 proteins for which a reliable 3D model could be obtained through standard modeling techniques. Despite showing poor sensitivity, this method can achieve a specificity of 0.70 and a positive predictive value of 0.29 by combining these two simple parameters. These values are slightly higher than those obtained with other established sequence-based or structure-based methods that have been evaluated using the same epitopes dataset. This method is implemented in a web server called B-Pred, which is accessible at http://immuno.bio.uniroma2.it/bpred. The server contains a number of original features that allow users to perform personalized reagent searches by manipulating the sliding window's width and sliding step, changing the exposure and model quality thresholds, and running sequential queries with different parameters. The B-Pred server should assist experimentalists in the rational selection of epitope antigens for a wide range of applications.
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http://dx.doi.org/10.2147/AABC.S30620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413014PMC
October 2012

A new Mycobacterium tuberculosis smooth colony reduces growth inside human macrophages and represses PDIM Operon gene expression. Does an heterogeneous population exist in intracellular mycobacteria?

Microb Pathog 2012 Sep 4;53(3-4):135-46. Epub 2012 Jul 4.

Institute of Cell Biology and Neurobiology, National Research Council (IBCN_CNR), Via E. Ramarini 32, 00016 Monterotondo Scalo, Rome, Italy.

Mycobacterium tuberculosis (MTB) colony morphology was associated to the pathogen's virulence. We isolated a new MTB H37Rv smooth colony, which only appeared following human macrophages (MDM) infection. The new phenotype was Alcohol-Acid resistant, but devoid of a covering capsule and biofilm defective. We ascertained that there were no deletions in the Rv0096-Rv0101 PDIM Operon, but that its expression was repressed as compared to MTB wild type (wt). Its lipid composition displayed lower PDIM components and higher TAG as compared to wt. In MDM it induced the sigma factors sigB, sigI and sigL expression vs. synthetic medium culture, while it repressed other six sigma factors. It also induced, significantly more than wt, mprA, a mycobacterial persistence regulator. It was phagocytosed more than wt by MDM, where it grew significantly less, but persisted therein till 14 days infection. It induced significantly less IFN-γ, IL-12 and IL-27 transcription than wt in infected MDM, while it increased the transcription of inducible NOS. It resided in mature, LAMP-3 positive phagolysosomes, where it never formed cords. This apparently "weaker" colony might represent an adaptive intracellular phenotype, whose infection may be less productive, but probably better equipped for a long lasting persistence in mildly activated host cells.
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http://dx.doi.org/10.1016/j.micpath.2012.06.002DOI Listing
September 2012

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection.

Proc Natl Acad Sci U S A 2012 May 25;109(21):E1360-8. Epub 2012 Apr 25.

Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy.

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1β, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-β without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1β, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.
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http://dx.doi.org/10.1073/pnas.1200484109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361443PMC
May 2012

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis.

Cell Microbiol 2012 Mar 13;14(3):356-67. Epub 2011 Dec 13.

Institutes of Microbiology Physics, Universita' Cattolica del Sacro Cuore, Rome, Italy.

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01721.xDOI Listing
March 2012

Lysophosphatidic acid enhances antimycobacterial response during in vivo primary Mycobacterium tuberculosis infection.

Cell Immunol 2011 1;271(1):1-4. Epub 2011 Jun 1.

Institute of Microbiology, Catholic University of the Sacred Heart, 00168 Rome, Italy.

Lysophospholipids may play an important protective role during primary infection of Mycobacterium tuberculosis (MTB) by enhancing innate antimycobacterial immune response of both macrophages and alveolar epithelial cells. Here, we show that treatment with lysophosphatidic acid (LPA) of mice aerogenically infected with MTB immediately after infection results in a significant early reduction of pulmonary CFUs and of histopathological damage in comparison with control mice. In contrast, treatment of acute disease does not result in any improvement of both microbiological and histopathological parameters. Altogether, these results show that LPA treatment can exert protective effect if administrated during primary infection, only.
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http://dx.doi.org/10.1016/j.cellimm.2011.05.014DOI Listing
December 2011

Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells.

Immunology 2010 Jan 22;129(1):125-32. Epub 2009 Jun 22.

Department of Biology, University of Rome Tor Vergata, Rome, Italy.

Human alveolar epithelial cells actively contribute to the innate immune response in the lung and play an important role in mycobacterial dissemination during primary infection, by undergoing cell death and by releasing mycobacteria. In the present study, we report that natural lysophospholipids, such as lysophosphatidic acid or sphingosine 1-phosphate, reduce Mycobacterium tuberculosis-induced cytotoxicity and enhance anti-mycobacterial activity in the A549 cell line, used as a model of type II alveolar epithelial cells. Intracellular mycobacterial killing was strictly dependent on phagolysosome maturation, which in turn was promoted by the activation of a Ca(2+)dependent phospholipase D. Finally, the restriction of mycobacteria in highly microbiocidal compartments was associated, in vitro, with a significant decrease in mycobacterial dissemination to macrophages. Taken as whole, these results suggest that the pulmonary lysophospholipid microenvironment may play a protective role during the early phases of host-pathogen interaction by enhancing anti-mycobacterial activity in type II alveolar epithelial cells.
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http://dx.doi.org/10.1111/j.1365-2567.2009.03145.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807493PMC
January 2010

CpG oligodeoxynucleotides promote phospholipase D dependent phagolysosome maturation and intracellular mycobacterial killing in M. tuberculosis infected type II alveolar epithelial cells.

Cell Immunol 2009 6;259(1):1-4. Epub 2009 Jun 6.

Department of Biology, University of Rome Tor Vergata, Rome, Italy.

CpG oligodeoxynucleotides have been previously shown to enhance antimycobacterial response in human monocytes/macrophages. The present study reports evidences showing the capability of CpG oligodeoxynucleotides to induce (i) host phospholipase D (PLD) activation, (ii) PLD dependent reactive oxygen intermediate production, (iii) PLD dependent phagolysosome maturation and (iv) PLD dependent intracellular mycobacterial killing in type II alveolar epithelial cells. These are the first evidences showing that alveolar epithelial cells may represent efficient effecter cells during primary innate antimycobacterial immune response.
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http://dx.doi.org/10.1016/j.cellimm.2009.06.002DOI Listing
September 2009

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes.

Biochem Biophys Res Commun 2007 Sep 25;361(3):687-93. Epub 2007 Jul 25.

Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.

Sphingosine 1-phosphate (S1P) has recently been described to induce antimycobacterial activity. The present study analyses the role played by S1P in antigen presentation of monocytes and in the next activation of Mycobacterium tuberculosis (MTB)-specific CD4+ T cell response. Results reported herein show that S1P stimulation of MTB-infected monocytes (i) inhibits intracellular mycobacterial growth, (ii) enhances phagolysosome maturation and the transit of mycobacteria in MHC class II compartments, (iii) increases the frequency of MTB-specific CD4+CD69+ T cells, expressing the inflammatory homing receptor CCR5, derived from tuberculosis patients and PPD+, BCG naïve, healthy subjects, and (iv) induces IFN-gamma production in CD4+CD69+CCR5+ T cells derived from PPD+ healthy individuals, only. Altogether, these results show that S1P promotes antigen processing and presentation in monocytes, increases the frequency of MTB-specific CD4+ T cells and can regulate IFN-gamma production by antigen specific CD4+ T cells in the course of active disease.
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http://dx.doi.org/10.1016/j.bbrc.2007.07.087DOI Listing
September 2007

Does sphingosine 1-phosphate play a protective role in the course of pulmonary tuberculosis?

Clin Immunol 2006 Dec 16;121(3):260-4. Epub 2006 Oct 16.

Department of Biology, University of Rome Tor Vergata Via della Ricerca Scientifica-00133, Rome, Italy.

Sphingosine 1-phosphate (S1P) has recently been reported to induce antimycobacterial activity in vitro and in a mouse model of in vivo Mycobacterium tuberculosis infection. However, its role in the course of pulmonary tuberculosis in humans is still not known. This study shows that S1P levels in airway surface fluid of tuberculosis (TB) patients are significantly less than those observed in non-TB control patients. Moreover, the in vitro stimulation of bronchoalveolar lavage cells coming from TB patients with S1P significantly reduces intracellular growth of endogenous mycobacterial isolates. These results show that, in the course of pulmonary TB, airway epithelial fluid-associated S1P may play a protective role in the containment of intracellular mycobacterial growth and that its decrease may represent a novel pathogenic mechanism through which M. tuberculosis favors its replication.
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http://dx.doi.org/10.1016/j.clim.2006.09.002DOI Listing
December 2006

CpG oligodeoxynucleotides induce Ca2+-dependent phospholipase D activity leading to phagolysosome maturation and intracellular mycobacterial growth inhibition in monocytes.

Biochem Biophys Res Commun 2006 Sep 18;347(4):963-9. Epub 2006 Jul 18.

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) have been reported to induce antimycobacterial activity both in vitro and in vivo. The present study analyzes the signals leading to CpG ODN-induced antimicrobial activity in monocytes. In this context, CpG, but not GpC, ODN induced cytosolic Ca2+ influx of extracellular origin which, in turn, activated host phospholipase D (PLD). The production of CpG-induced PLD-dependent phosphatidic acid induced the maturation of phagolysosomes and intracellular mycobacterial growth inhibition. These results show the presence of an antimicrobial pathway in monocytes, mediated by Ca2+-dependent PLD which can be useful for the exploitation of novel anti-tuberculosis immunotherapy approaches.
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http://dx.doi.org/10.1016/j.bbrc.2006.06.186DOI Listing
September 2006