Publications by authors named "Lucia Nencioni"

53 Publications

System-oriented optimization of multi-target 2,6-diaminopurine derivatives: Easily accessible broad-spectrum antivirals active against flaviviruses, influenza virus and SARS-CoV-2.

Eur J Med Chem 2021 Jul 5;224:113683. Epub 2021 Jul 5.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Viale delle Scienze, 27/A, 43124, Parma, Italy. Electronic address:

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC = 0.5-5.3 μM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC = 0.5 μM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets.
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http://dx.doi.org/10.1016/j.ejmech.2021.113683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255191PMC
July 2021

Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models.

Int J Mol Sci 2021 Jun 11;22(12). Epub 2021 Jun 11.

Institute of Translational Pharmacology, National Research Council (CNR), 00133 Rome, Italy.

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer's disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.
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http://dx.doi.org/10.3390/ijms22126279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230621PMC
June 2021

Intracellular Redox-Modulated Pathways as Targets for Effective Approaches in the Treatment of Viral Infection.

Int J Mol Sci 2021 Mar 30;22(7). Epub 2021 Mar 30.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via Saffi 2, 61029 Urbino (PU), Italy.

Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-B, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-B, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses.
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http://dx.doi.org/10.3390/ijms22073603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036776PMC
March 2021

Investigation of (Nees) Engl. Oil and Its Main Components for Antiviral Activity.

Pharmaceuticals (Basel) 2021 Mar 9;14(3). Epub 2021 Mar 9.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, "Sapienza" Università di Roma, p.le Aldo Moro 5, I-00185 Rome, Italy.

The resinous exudate produced by (Nees) Engl. is commonly known as true myrrh and has been used since antiquity for several medicinal applications. Hundreds of metabolites have been identified in the volatile component of myrrh so far, mainly sesquiterpenes. Although several efforts have been devoted to identifying these sesquiterpenes, the phytochemical analyses have been performed by gas-chromatography/mass spectrometry (GC-MS) where the high temperature employed can promote degradation of the components. In this work, we report the extraction of by supercritical CO, an extraction method known for the mild extraction conditions that allow avoiding undesired chemical reactions during the process. In addition, the analyses of myrrh oil and of its metabolites were performed by HPLC and GC-MS. Moreover, we evaluated the antiviral activity against influenza A virus of the myrrh extracts, that was possible to appreciate after the addition of vitamin E acetate (α-tocopheryl acetate) to the extract. Further, the single main bioactive components of the oil of commercially available were tested. Interestingly, we found that both furanodienone and curzerene affect viral replication by acting on different steps of the virus life cycle.
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http://dx.doi.org/10.3390/ph14030243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999460PMC
March 2021

Laccase-Catalyzed 1,4-Dioxane-Mediated Synthesis of Belladine -Oxides with Anti-Influenza A Virus Activity.

Int J Mol Sci 2021 Jan 29;22(3). Epub 2021 Jan 29.

Department of Ecology and Biology, University of Tuscia, 01100 Viterbo, Italy.

Belladine -oxides active against influenza A virus have been synthetized by a novel laccase-catalyzed 1,4-dioxane-mediated oxidation of aromatic and side-chain modified belladine derivatives. Electron paramagnetic resonance (EPR) analysis confirmed the role of 1,4-dioxane as a co-oxidant. The reaction was chemo-selective, showing a high functional-group compatibility. The novel belladine -oxides were active against influenza A virus, involving the early stage of the virus replication life cycle.
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http://dx.doi.org/10.3390/ijms22031337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866262PMC
January 2021

Redox-Modulating Agents in the Treatment of Viral Infections.

Int J Mol Sci 2020 Jun 8;21(11). Epub 2020 Jun 8.

Department of Public Health and Infectious Diseases, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, 00185 Rome, Italy.

Viruses use cell machinery to replicate their genome and produce viral proteins. For this reason, several intracellular factors, including the redox state, might directly or indirectly affect the progression and outcome of viral infection. In physiological conditions, the redox balance between oxidant and antioxidant species is maintained by enzymatic and non-enzymatic systems, and it finely regulates several cell functions. Different viruses break this equilibrium and induce an oxidative stress that in turn facilitates specific steps of the virus lifecycle and activates an inflammatory response. In this context, many studies highlighted the importance of redox-sensitive pathways as novel cell-based targets for therapies aimed at blocking both viral replication and virus-induced inflammation. In the review, we discuss the most recent findings in this field. In particular, we describe the effects of natural or synthetic redox-modulating molecules in inhibiting DNA or RNA virus replication as well as inflammatory pathways. The importance of the antioxidant transcription factor Nrf2 is also discussed. Most of the data reported here are on influenza virus infection. We believe that this approach could be usefully applied to fight other acute respiratory viral infections characterized by a strong inflammatory response, like COVID-19.
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http://dx.doi.org/10.3390/ijms21114084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312898PMC
June 2020

Glutathione increase by the n-butanoyl glutathione derivative (GSH-C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus.

FASEB Bioadv 2019 May 13;1(5):296-305. Epub 2019 Mar 13.

Deparment of Public Health and Infectious Diseases Istituto Pasteur Italia-Fondazione Cenci-Bolognetti, Sapienza University of Rome Rome Italy.

During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N-butanoyl GSH derivative (GSH-C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH-C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH-C4. Moreover, the treatment with GSH-C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH-C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile.
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http://dx.doi.org/10.1096/fba.2018-00066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996388PMC
May 2019

Role of Glutathionylation in Infection and Inflammation.

Nutrients 2019 Aug 20;11(8). Epub 2019 Aug 20.

IRCCS San Raffaele Pisana, Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, 00166 Rome, Italy.

Glutathionylation, that is, the formation of mixed disulfides between protein cysteines and glutathione (GSH) cysteines, is a reversible post-translational modification catalyzed by different cellular oxidoreductases, by which the redox state of the cell modulates protein function. So far, most studies on the identification of glutathionylated proteins have focused on cellular proteins, including proteins involved in host response to infection, but there is a growing number of reports showing that microbial proteins also undergo glutathionylation, with modification of their characteristics and functions. In the present review, we highlight the signaling role of GSH through glutathionylation, particularly focusing on microbial (viral and bacterial) glutathionylated proteins (GSSPs) and host GSSPs involved in the immune/inflammatory response to infection; moreover, we discuss the biological role of the process in microbial infections and related host responses.
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http://dx.doi.org/10.3390/nu11081952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723385PMC
August 2019

Erratum to "Counteraction of HCV-Induced Oxidative Stress Concurs to Establish Chronic Infection in Liver Cell Cultures".

Oxid Med Cell Longev 2019 18;2019:3712969. Epub 2019 Apr 18.

Istituto Superiore di Sanità, Center for Gender-Specific Medicine, Rome, Italy.

[This corrects the article DOI: 10.1155/2019/6452390.].
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http://dx.doi.org/10.1155/2019/3712969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501126PMC
April 2019

Counteraction of HCV-Induced Oxidative Stress Concurs to Establish Chronic Infection in Liver Cell Cultures.

Oxid Med Cell Longev 2019 13;2019:6452390. Epub 2019 Feb 13.

Istituto Superiore di Sanità, Center for Gender-Specific Medicine, Rome, Italy.

Hepatitis C virus (HCV) is a blood-borne pathogen causing acute and chronic hepatitis. A significant number of people chronically infected with HCV develop cirrhosis and/or liver cancer. The pathophysiologic mechanisms of hepatocyte damage associated with chronic HCV infection are not fully understood yet, mainly due to the lack of an system able to recapitulate the stages of infection . Several studies underline that HCV virus replication depends on redox-sensitive cellular pathways; in addition, it is known that virus itself induces alterations of the cellular redox state. However, the exact interplay between HCV replication and oxidative stress has not been elucidated. In particular, the role of reduced glutathione (GSH) in HCV replication and infection is still not clear. We set up an system, based on low m.o.i. of Huh7.5 cell line with a HCV infectious clone (J6/JFH1), that reproduced the acute and persistent phases of HCV infection up to 76 days of culture. We demonstrated that the acute phase of HCV infection is characterized by the elevated levels of reactive oxygen species (ROS) associated in part with an increase of NADPH-oxidase transcripts and activity and a depletion of GSH accompanied by high rates of viral replication and apoptotic cell death. Conversely, the chronic phase is characterized by a reestablishment of reduced environment due to a decreased ROS production and increased GSH content in infected cells that might concur to the establishment of viral persistence. Treatment with the prooxidant auranofin of the persistently infected cultures induced the increase of viral RNA titer, suggesting that a prooxidant state could favor the reactivation of HCV viral replication that in turn caused cell damage and death. Our results suggest that targeting the redox-sensitive host-cells pathways essential for viral replication and/or persistence may represent a promising option for contrasting HCV infection.
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http://dx.doi.org/10.1155/2019/6452390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393922PMC
May 2019

Redox alteration in patients infected by different HCV genotypes.

Infez Med 2018 Sep;26(3):249-254

Laboratory of Molecular Virology, Polyclinic Tor Vergata Foundation, Rome, Italy.

Chronic hepatitis C virus (HCV) infection plays a pivotal role in hepatocarcinogenesis and has been associated with oxidative DNA damage. Few data have been reported on the general redox state in patients infected with different HCV genotypes. Total antioxidant capacity (TAC) and hydrogen peroxide levels as well as oxidative stress index were measured in serum of hepatitis C chronic patients in relation to genotype, viral load, transaminases level and degree of fibrosis. Serum was obtained from two-hundred-fifty-two HCV infected patients and twenty-five healthy donors. TAC was measured by TAC Colorimetric Assay and hydrogen peroxide concentration by Hydrogen Peroxide Colorimetric Assay Kit. In HCV infected patients, mean serum TAC was 5.62 mM Trolox equivalents which was significantly lower (p < 0.0001) than control group (7.25 mM Trolox equivalents). TAC reduction was particularly evident in patients infected by genotype 2 compared to those infected by genotypes 1, 3 and 4. In parallel, high levels of hydrogen peroxide were found in the serum of infected patients, p=0.0015. Although no statistically significant correlation was found with the degree of fibrosis, transaminases level or viral load, oxidative stress index was higher in HCV infected patients compared to uninfected controls, p=0.003. The results indicate an imbalance of the redox state in HCV infected patients, with a strong reduction of the total antioxidant capacity and high oxidative stress index. Because oxidative burden may favour disease progression, a novel strategy aimed at counteracting it by using antioxidant molecules as adjunct therapy might represent a useful tool in the management of HCV chronic infection.
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September 2018

Antiviral and Antioxidant Activity of a Hydroalcoholic Extract from L.

Oxid Med Cell Longev 2018 24;2018:5919237. Epub 2018 Jul 24.

Department of Public Health and Infectious Diseases, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.

A hydroalcoholic extract from female inflorescences of L. (HOP extract) was evaluated for its anti-influenza activity. The ability of the extract to interfere with different phases of viral replication was assessed, as well as its effect on the intracellular redox state, being unbalanced versus the oxidative state in infected cells. The radical scavenging power, inhibition of lipoperoxidation, and ferric reducing activity were assayed as antioxidant mechanisms. A phytochemical characterization of the extract was also performed. We found that HOP extract significantly inhibited replication of various viral strains, at different time from infection. Viral replication was partly inhibited when virus was incubated with extract before infection, suggesting a direct effect on the virions. Since HOP extract was able to restore the reducing conditions of infected cells, by increasing glutathione content, its antiviral activity might be also due to an interference with redox-sensitive pathways required for viral replication. Accordingly, the extract exerted radical scavenging and reducing effects and inhibited lipoperoxidation and the tBOOH-induced cytotoxicity. At phytochemical analysis, different phenolics were identified, which altogether might contribute to HOP antiviral effect. In conclusion, our results highlighted anti-influenza and antioxidant properties of HOP extract, which encourage further studies to evaluate its possible application.
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http://dx.doi.org/10.1155/2018/5919237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081516PMC
October 2018

A Polyphenol Rich Extract from L. DR2 Peel Exhibits Antioxidant Properties and Anti-Herpes Simplex Virus Type 1 Activity In Vitro.

Molecules 2018 Aug 17;23(8). Epub 2018 Aug 17.

Department of Public Health and Infectious Diseases, Istituto Pasteur Italia-Fondazione Cenci-Bolognetti, Sapienza University, P.le Aldo Moro 5, 00185 Rome, Italy.

DR2B and DR2C extracts, obtained by ethanolic maceration of peel from commercially and physiologically ripe aubergine berries, were studied for the antioxidative cytoprotective properties and anti-HSV-1 activity, in line with the evidence that several antioxidants can impair viral replication by maintaining reducing conditions in host cells. The antioxidative cytoprotective effects against tBOOH-induced damage were assessed in Caco2 cells, while antiviral activity was studied in Vero cells; polyphenolic fingerprints were characterized by integrated phytochemical methods. Results highlighted different compositions of the extracts, with chlorogenic acid and delphinidin-3-rutinoside as the major constituents; other peculiar phytochemicals were also identified. Both samples reduced reactive oxygen species (ROS) production and exhibited scavenging and chelating properties. DR2C partly counteracted the tBOOH-induced cytotoxicity, with a remarkable lowering of lactate metabolism under both normoxia and hypoxia; interestingly, it increased intracellular GSH levels. Furthermore, DR2C inhibited the HSV-1 replication when added for 24 h after viral adsorption, as also confirmed by the reduction of many viral proteins' expression. Since DR2C was able to reduce NOX4 expression during HSV-1 infection, its antiviral activity may be correlated to its antioxidant properties. Although further studies are needed to better characterize DR2C activity, the results suggest this extract as a promising new anti-HSV-1 agent.
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http://dx.doi.org/10.3390/molecules23082066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222547PMC
August 2018

Differential Redox State Contributes to Sex Disparities in the Response to Influenza Virus Infection in Male and Female Mice.

Front Immunol 2018 30;9:1747. Epub 2018 Jul 30.

Department of Public Health and Infectious Diseases, Pasteur Institute Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.
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http://dx.doi.org/10.3389/fimmu.2018.01747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077261PMC
September 2019

Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with Antioxidant and Anti-influenza Activities.

J Nat Prod 2017 12 13;80(12):3247-3254. Epub 2017 Dec 13.

Department of Ecology and Biology, University of Tuscia , Via C. De Lellis, Viterbo, 01100, Italy.

Different catechol and pyrogallol derivatives have been synthesized by oxidation of coumarins with 2-iodoxybenzoic acid (IBX) in DMSO at 25 °C. A high regioselectivity was observed in accordance with the stability order of the incipient carbocation or radical benzylic-like intermediate. The oxidation was also effective in water under heterogeneous conditions by using IBX supported on polystyrene. The new derivatives showed improved antioxidant effects in the DPPH test and inhibitory activity against the influenza A/PR8/H1N1 virus. These data represent a new entry for highly oxidized coumarins showing an antiviral activity possibly based on the control of the intracellular redox value.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00665DOI Listing
December 2017

MC1568 inhibits HDAC6/8 activity and influenza A virus replication in lung epithelial cells: role of Hsp90 acetylation.

Future Med Chem 2016 Nov 14;8(17):2017-2031. Epub 2016 Oct 14.

Department of Public Health & Infectious Diseases, 'Sapienza' University of Rome, P.le A. Moro 5, 00185 Rome, Italy.

Aim: Histone deacetylases (HDACs) regulate the life cycle of several viruses. We investigated the ability of different HDAC inhibitors, to interfere with influenza virus A/Puerto Rico/8/34/H1N1 (PR8 virus) replication in Madin-Darby canine kidney and NCI cells.

Results: 3-(5-(3-Fluorophenyl)-3-oxoprop-1-en-1-yl)-1-methyl-1H-pyrrol-2-yl)-N-hydroxyacrylamide (MC1568) inhibited HDAC6/8 activity and PR8 virus replication, with decreased expression of viral proteins and their mRNAs. Such an effect may be related to a decrease in intranuclear content of viral polymerases and, in turn, to an early acetylation of Hsp90, a major player in their nuclear import. Later, the virus itself induced Hsp90 acetylation, suggesting a differential and time-dependent role of acetylated proteins in virus replication.

Conclusion: The inhibition of HDAC6/8 activity during early steps of PR8 virus replication could lead to novel anti-influenza strategy.
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http://dx.doi.org/10.4155/fmc-2016-0073DOI Listing
November 2016

Validation of a Reversed-Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs.

Oxid Med Cell Longev 2016 17;2016:1746985. Epub 2016 Jan 17.

Department of Biomolecular Sciences, University of Urbino "Carlo Bo", 61029 Urbino, Italy.

A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313 μM and 1.25 μM for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains.
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http://dx.doi.org/10.1155/2016/1746985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739232PMC
December 2016

Carbon nanotubes supported tyrosinase in the synthesis of lipophilic hydroxytyrosol and dihydrocaffeoyl catechols with antiviral activity against DNA and RNA viruses.

Bioorg Med Chem 2015 Sep 30;23(17):5345-51. Epub 2015 Jul 30.

Department of Ecology and Biology, University of Tuscia, Largo dell'Università, 01100 Viterbo (VT), Italy. Electronic address:

Hydroxytyrosol and dihydrocaffeoyl catechols with lipophilic properties have been synthesized in high yield using tyrosinase immobilized on multi-walled carbon nanotubes by the Layer-by-Layer technique. All synthesized catechols were evaluated against a large panel of DNA and RNA viruses, including Poliovirus type 1, Echovirus type 9, Herpes simplex virus type 1 (HSV-1), Herpes simplex virus type 2 (HSV-2), Coxsackievirus type B3 (Cox B3), Adenovirus type 2 and type 5 and Cytomegalovirus (CMV). A significant antiviral activity was observed in the inhibition of HSV-1, HSV-2, Cox B3 and CMV. The mechanism of action of the most active dihydrocaffeoyl derivative was investigated against a model of HSV-1 infection.
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http://dx.doi.org/10.1016/j.bmc.2015.07.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125559PMC
September 2015

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress.

PLoS One 2015 18;10(5):e0127086. Epub 2015 May 18.

Brighton & Sussex Medical School, Falmer, Brighton, United Kingdom.

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127086PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436175PMC
April 2016

Influenza A virus infection of intestinal epithelial cells enhances the adhesion ability of Crohn's disease associated Escherichia coli strains.

PLoS One 2015 23;10(2):e0117005. Epub 2015 Feb 23.

Department of Public Health and Infectious Diseases, "Sapienza" University, Rome, Italy.

Modifications of intestinal glycoreceptors expression, in particular CEACAM6, typically found in ileal Crohn's disease (CD), favor, among the commensal species of microbiota, the enrichment in Escherichia coli. Removal of protein glycosidic residues by neuraminidase, a sialidase typical of influenza virus, increases adhesion ability of Escherichia coli to Caco-2 intestinal cells. In this study we investigated whether influenza virus infection of human intestinal epithelial cells could influence the adhesiveness of different Escherichia coli strains isolated from CD patients by altering surface glycoreceptors. Influenza virus infection of intestinal cells increased exposure of galactose and mannose residues on the cell surface. In particular, glycoreceptors Thomsen-Friedenreich and CEACAM6 were over-expressed in influenza virus infected cells. In the same experimental conditions, a significant increase in bacterial adhesiveness was observed, independently of their own adhesive ability. The increase was reverted by treatment with anti-TF and anti-CEACAM6 antibodies. Interestingly, influenza virus was able to efficiently replicate in human primary intestinal cells leading to TF exposure. Finally, intestinal infected cells produced high levels of pro-inflammatory cytokines compared to control. Overall these data suggest that influenza virus infection, could constitute an additional risk factor in CD patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117005PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338238PMC
January 2016

New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis C virus replication via suppression of cyclooxygenase-2.

Eur J Med Chem 2015 Jan 27;90:497-506. Epub 2014 Nov 27.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy. Electronic address:

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 μM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 μM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 μM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 μM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels.
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http://dx.doi.org/10.1016/j.ejmech.2014.11.042DOI Listing
January 2015

Intracellular redox state as target for anti-influenza therapy: are antioxidants always effective?

Curr Top Med Chem 2014 ;14(22):2529-41

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy.

Influenza virus infections represent a big issue for public health since effective treatments are still lacking. In particular, the emergence of strains resistant to drugs limits the effectiveness of anti-influenza agents. For this reason, many efforts have been dedicated to the identification of new therapeutic strategies aimed at targeting the virus-host cell interactions. Oxidative stress is a characteristic of some viral infections including influenza. Because antioxidants defend cells from damage caused by reactive oxygen species induced by different stimuli including pathogens, they represent interesting molecules to fight infectious diseases. However, most of the available studies have found that these would-be panaceas could actually exacerbate the diseases they claim to prevent, and have thus revealed "the dark side" of these molecules. This review article discusses the latest opportunities and drawbacks of the antioxidants used in anti-influenza therapy and new perspectives.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435240PMC
http://dx.doi.org/10.2174/1568026614666141203125211DOI Listing
September 2015

Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by NOX4-derived ROS.

Cell Microbiol 2015 Jan 7;17(1):131-45. Epub 2014 Oct 7.

Department of Public Health and Infectious Diseases, Pasteur Institute-Fondazione Cenci-Bolognetti, Sapienza University of Rome, Rome, 00185, Italy; CEINGE Advanced Biotechnology, Naples, 80145, Italy.

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus-induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4-derived ROS in activating redox-regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4-mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.
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http://dx.doi.org/10.1111/cmi.12343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311438PMC
January 2015

Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.

Front Microbiol 2014 5;5:280. Epub 2014 Jun 5.

Microbiology Section, Department of Public Health and Infectious Diseases, "Sapienza" University Rome, Italy.

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.
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http://dx.doi.org/10.3389/fmicb.2014.00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046265PMC
June 2014

In vitro inhibition of herpes simplex virus type 1 replication by Mentha suaveolens essential oil and its main component piperitenone oxide.

Phytomedicine 2014 May 11;21(6):857-65. Epub 2014 Mar 11.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy. Electronic address:

Several essential oils exert in vitro activity against bacteria and viruses and, among these latter, herpes simplex virus type 1 (HSV-1) is known to develop resistance to commonly used antiviral agents. Thus, the effects of the essential oil derived from Mentha suaveolens (EOMS) and its active principle piperitenone oxide (PEO) were tested in in vitro experimental model of infection with HSV-1. The 50% inhibitory concentration (IC50) was determined at 5.1μg/ml and 1.4μg/ml for EOMS and PEO, respectively. Australian tea tree oil (TTO) was used as control, revealing an IC50 of 13.2μg/ml. Moreover, a synergistic action against HSV-1 was observed when each oil was added in combination with acyclovir. In order to find out the mechanism of action, EOMS, PEO and TTO were added to the cells at different times during the virus life-cycle. Results obtained by yield reduction assay indicated that the antiviral activity of both compounds was principally due to an effect after viral adsorption. Indeed, no reduction of virus yield was observed when cells were treated during viral adsorption or pre-treated before viral infection. In particular, PEO exerted a strong inhibitory effect by interfering with a late step of HSV-1 life-cycle. HSV-1 infection is known to induce a pro-oxidative state with depletion of the main intracellular antioxidant glutathione and this redox change in the cell is important for viral replication. Interestingly, the treatment with PEO corrected this deficit, thus suggesting that the compound could interfere with some redox-sensitive cellular pathways exploited for viral replication. Overall our data suggest that both EOMS and PEO could be considered good candidates for novel anti-HSV-1 strategies, and need further exploration to better characterize the targets underlying their inhibition.
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http://dx.doi.org/10.1016/j.phymed.2014.01.013DOI Listing
May 2014

Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole.

PLoS One 2014 27;9(2):e90230. Epub 2014 Feb 27.

Dipartimento di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio", Chieti, Italy.

Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183)PAPAP(187) motif of OmpA, nor the N-terminal polyproline (43)PPPP(46) motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090230PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937361PMC
October 2014

Tyrosinase and Layer-by-Layer supported tyrosinases in the synthesis of lipophilic catechols with antiinfluenza activity.

Bioorg Med Chem 2013 Dec 30;21(24):7699-708. Epub 2013 Oct 30.

Department of Ecology and Biology, University of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy.

Catechol derivatives with lipophilic properties have been selectively synthesized by tyrosinase in high yield avoiding long and tedious protection/deprotection steps usually required in traditional procedures. The synthesis was effective also with immobilized tyrosinase able to perform for more runs. The novel catechols were evaluated against influenza A virus, that continue to represent a severe threat worldwide. A significant antiviral activity was observed in derivatives characterized by antioxidant activity and long carbon alkyl side-chains, suggesting the possibility of a new inhibition mechanism based on both redox and lipophilic properties.
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http://dx.doi.org/10.1016/j.bmc.2013.10.026DOI Listing
December 2013

Human Polyomavirus JC monitoring and noncoding control region analysis in dynamic cohorts of individuals affected by immune-mediated diseases under treatment with biologics: an observational study.

Virol J 2013 Sep 30;10:298. Epub 2013 Sep 30.

Department of Public Health and Infectious Diseases, Sapienza University, P,le Aldo Moro, 5, 00185 Rome, Italy.

Background: Progressive multifocal leukoencephalopathy (PML) onset, caused by Polyomavirus JC (JCPyV) in patients affected by immune-mediated diseases during biological treatment, raised concerns about the safety profile of these agents. Therefore, the aims of this study were the JCPyV reactivation monitoring and the noncoding control region (NCCR) and viral protein 1 (VP1) analysis in patients affected by different immune-mediated diseases and treated with biologics.

Methods: We performed JCPyV-specific quantitative PCR of biological samples collected at moment of recruitment (t0) and every 4 months (t1, t2, t3, t4). Subsequently, rearrangements' analysis of NCCR and VP1 was carried out. Data were analyzed using χ2 test.

Results: Results showed that at t0 patients with chronic inflammatory rheumatic diseases presented a JCPyV load in the urine significantly higher (p≤0.05) than in patients with multiple sclerosis (MS) and Crohn's disease (CD). It can also be observed a significant association between JC viruria and JCPyV antibodies after 1 year of natalizumab (p=0.04) in MS patients. Finally, NCCR analysis showed the presence of an archetype-like sequence in all urine samples, whereas a rearranged NCCR Type IR was found in colon-rectal biopsies collected from 2 CD patients after 16 months of infliximab. Furthermore, sequences isolated from peripheral blood mononuclear cells (PBMCs) of 2 MS patients with JCPyV antibody at t0 and t3, showed a NCCR Type IIR with a duplication of a 98 bp unit and a 66 bp insert, resulting in a boxB deletion and 37 T to G transversion into the Spi-B binding site. In all patients, a prevalence of genotypes 1A and 1B, the predominant JCPyV genotypes in Europe, was observed.

Conclusions: It has been important to understand whether the specific inflammatory scenario in different immune-mediated diseases could affect JCPyV reactivation from latency, in particular from kidneys. Moreover, for a more accurate PML risk stratification, testing JC viruria seems to be useful to identify patients who harbor JCPyV but with an undetectable JCPyV-specific humoral immune response. In these patients, it may also be important to study the JCPyV NCCR rearrangement: in particular, Spi-B expression in PBMCs could play a crucial role in JCPyV replication and NCCR rearrangement.
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http://dx.doi.org/10.1186/1743-422X-10-298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849738PMC
September 2013

New insights on human polyomavirus JC and pathogenesis of progressive multifocal leukoencephalopathy.

Clin Dev Immunol 2013 17;2013:839719. Epub 2013 Apr 17.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, P.le Aldo Moro, 5-00185 Rome, Italy.

John Cunningham virus (JCV) is a member of the Polyomaviridae family. It was first isolated from the brain of a patient with Hodgkin disease in 1971, and since then the etiological agent of the progressive multifocal leukoencephalopathy (PML) was considered. Until the human immunodeficiency virus (HIV) pandemic, PML was rare: in fact HIV-induced immunodeficiency is the most common predisposing factor accounting for 85% of all instances of PML. This data led to intense research on JCV infection and resulted in better understanding of epidemiology and clinic-pathologic spectrum. Recently, cases of PML have been observed after the introduction of monoclonal antibodies, such as natalizumab, rituximab, efalizumab, and infliximab, in the treatment of autoimmune disease, underlining the important role of host immunity in PML pathogenesis. In this review current understanding of the JCV infection and the new findings relating to the pathogenesis of PML has been comprehensively revised, focusing our attention on the interaction between the cellular and viral molecular pathways implicated in the JCV infection and the modulating role of host immune surveillance in the viral reactivation from a latent state.
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http://dx.doi.org/10.1155/2013/839719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652120PMC
December 2013

Higher prevalence and abundance of Bdellovibrio bacteriovorus in the human gut of healthy subjects.

PLoS One 2013 16;8(4):e61608. Epub 2013 Apr 16.

Department of Public Health and Infectious Diseases, 'Sapienza' University of Rome, Rome, Italy.

Introduction: Members of the human intestinal microbiota are key players in maintaining human health. Alterations in the composition of gut microbial community (dysbiosis) have been linked with important human diseases. Understanding the underlying processes that control community structure, including the bacterial interactions within the microbiota itself, is essential. Bdellovibrio bacteriovorus is a gram-negative bacterium that preys other gram-negative species for survival, acting as a population-balancer. It was found in terrestrial/aquatic ecosystems, and in animal intestines, postulating its presence also in the human gut.

Methods: The present study was aimed to evaluate, by end-point PCR and qPCR, the presence of B. bacteriovorus in intestinal and faecal biopsy specimens from 92 paediatric healthy subjects and patients, suffering from Inflammatory Bowel Diseases (IBD), Celiac disease and Cystic fibrosis (CF).

Results: i) B. bacteriovorus was present and abundant only in healthy individuals, while it was heavily reduced in patients, as in the case of IBD and Celiac, while in CF patients and relative controls we observed comparable results; ii) B. bacteriovorus seemed to be mucosa-associated, because all IBD and Celiac biopsies (and related controls) were treated with mucus-removing agents, leaving only the mucosa-attached microflora; iii) B. bacteriovorus abundance was district-dependent, with a major preponderance in duodenum, and gradually decreasing up to rectum; iv) B. bacteriovorus levels significantly dropped in disease status, in duodenum and ileum.

Conclusions: Results obtained in this study could represent the first step for new therapeutic strategies aimed to restore a balance in the intestinal ecosystem, utilizing Bdellovibrio as a probiotic.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061608PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628794PMC
November 2013
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