Publications by authors named "Daria Trabattoni"

116 Publications

Early-Transmitted Variants and Their Evolution in a HIV-1 Positive Couple: NGS and Phylogenetic Analyses.

Viruses 2021 03 19;13(3). Epub 2021 Mar 19.

Department of Biomedical and Clinical Sciences, University of Milan, Via G.B. Grassi 74, 20157 Milan, Italy.

We had access to both components of a couple who became infected with human immunodeficiency virus (HIV)-1 through sexual behavior during the early initial phase of infection and before initiation of therapy. We analyzed blood samples obtained at the time of diagnosis and after six months of combined antiretroviral therapy. Next-generation sequencing (NGS) and phylogenetic analyses were used to investigate the transmission and evolution of HIV-1 quasispecies. Phylogenetic analyses were conducted using Bayesian inference methods. Both partners were infected with an HIV-1 B subtype. No evidence of viral recombination was observed. The lowest intrapersonal genetic distances were observed at baseline, before initiation of therapy, and in particular in the V1V2 fragment (distances ranging from 0.102 to 0.148). One HIV-1 single variant was concluded to be dominant in all of the HIV-1 regions analyzed, although some minor variants could be observed. The same tree structure was observed both at baseline and after six months of therapy. These are the first extended phylogenetic analyses performed on both members of a therapy-naïve couple within a few weeks of infection, and in which the effect of antiretroviral therapy on viral evolution was analyzed. Understanding which HIV-1 variants are most likely to be transmitted would allow a better understanding of viral evolution, possibly playing a role in vaccine design and prevention strategies.
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http://dx.doi.org/10.3390/v13030513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003824PMC
March 2021

Emergency Lung Transplantation after COVID-19: Immunopathological Insights on Two Affected Patients.

Cells 2021 03 10;10(3). Epub 2021 Mar 10.

Division of Pathology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 20122 Milan, Italy.

We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs' GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.
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http://dx.doi.org/10.3390/cells10030611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999589PMC
March 2021

UV-C irradiation is highly effective in inactivating SARS-CoV-2 replication.

Sci Rep 2021 03 18;11(1):6260. Epub 2021 Mar 18.

Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.

The potential virucidal effects of UV-C irradiation on SARS-CoV-2 were experimentally evaluated for different illumination doses and virus concentrations (1000, 5, 0.05 MOI). At a virus density comparable to that observed in SARS-CoV-2 infection, an UV-C dose of just 3.7 mJ/cm was sufficient to achieve a more than 3-log inactivation without any sign of viral replication. Moreover, a complete inactivation at all viral concentrations was observed with 16.9 mJ/cm. These results could explain the epidemiological trends of COVID-19 and are important for the development of novel sterilizing methods to contain SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41598-021-85425-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973506PMC
March 2021

ERAPs Reduce In Vitro HIV Infection by Activating Innate Immune Response.

J Immunol 2021 Apr 22;206(7):1609-1617. Epub 2021 Feb 22.

Department of Biomedical and Clinical Sciences "L. Sacco," University of Milan, 20157 Milan, Italy;

Recombinant human (rh) ERAP2-treated PBMCs are less susceptible to in vitro HIV-1 infection even when CD8 T cells are depleted. We therefore investigated whether ERAP2 can trigger other immunocompetent cells, boosting their antiviral potential. To this end, human monocyte-derived macrophages (MDMs) differentiated from PBMCs of 15 healthy donors were in vitro HIV-1 infected in the presence/absence of 100 ng/ml of rhERAP2, rhERAP1, or rhERAP1+rhERAP2. Notably, rhERAP2 treatment resulted in a 7-fold reduction of HIV-1 replication in MDMs ( < 0.05). This antiviral activity was associated with an increased mRNA expression of CD80, IL-1β, IL-18, and TNF-α ( < 0.01 for cytokine) in in vitro ERAP2-treated HIV-1-infected MDMs and a greater release of IL-1β, TNF-α, IL-6, and IL-8 ( < 0.01 for each cytokine). The rhERAPs addition also induced the functional inflammasome activation by ASC speck formation in monocytes ( < 0.01) and in THP1-derived macrophages ( < 0.01) as well as a rise in the percentage of activated classical (CD14CD16HLA-DRIICCR7) and intermediate (CD14CD16HLA-DRIICCR7) monocytes ( < 0.02). Finally, THP-1-derived macrophages showed an increased phagocytosis following all ERAPs treatments. The discovery that ERAPs are able to trigger several antiviral mechanisms in monocyte/macrophages suggests that their anti-HIV potential is not limited to their canonical role in Ag presentation and CD8 T cell activation. These findings pose the premise to further investigate the role of ERAPs in both innate and adaptive immunostimulatory pathways and suggest their potential use in novel preventive and therapeutic approaches against HIV-1 infection.
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http://dx.doi.org/10.4049/jimmunol.2000991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980528PMC
April 2021

Mild SARS-CoV-2 Infection After Gene Therapy in a Child With Wiskott-Aldrich Syndrome: A Case Report.

Front Immunol 2020 24;11:603428. Epub 2020 Nov 24.

Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.

In this work we present the case of SARS-CoV-2 infection in a 1.5-year-old boy affected by severe Wiskott-Aldrich Syndrome with previous history of autoinflammatory disease, occurring 5 months after treatment with gene therapy. Before SARS-CoV-2 infection, the patient had obtained engraftment of gene corrected cells, resulting in WASP expression restoration and early immune reconstitution. The patient produced specific immunoglobulins to SARS-CoV-2 at high titer with neutralizing capacity and experienced a mild course of infection, with limited inflammatory complications, despite pre-gene therapy clinical phenotype.
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http://dx.doi.org/10.3389/fimmu.2020.603428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732473PMC
December 2020

Analysis of SARS-CoV-2 vertical transmission during pregnancy.

Nat Commun 2020 10 12;11(1):5128. Epub 2020 Oct 12.

Unit of Obstetrics and Gynecology, ASST Fatebenefratelli-Sacco, Department of Biological and Clinical Sciences, University of Milan, Milan, Italy.

The impact of SARS-CoV-2 infection during gestation remains unclear. Here, we analyse the viral genome on maternal and newborns nasopharyngeal swabs, vaginal swabs, maternal and umbilical cord plasma, placenta and umbilical cord biopsies, amniotic fluids and milk from 31 mothers with SARS-CoV-2 infection. In addition, we also test specific anti-SARS-CoV-2 antibodies and expression of genes involved in inflammatory responses in placentas, and in maternal and umbilical cord plasma. We detect SARS-CoV-2 genome in one umbilical cord blood and in two at-term placentas, in one vaginal mucosa and in one milk specimen. Furthermore, we report the presence of specific anti-SARS-CoV-2 IgM and IgG antibodies in one umbilical cord blood and in one milk specimen. Finally, in the three documented cases of vertical transmission, SARS-CoV-2 infection was accompanied by a strong inflammatory response. Together, these data support the hypothesis that in utero SARS-CoV-2 vertical transmission, while low, is possible. These results might help defining proper obstetric management of COVID-19 pregnant women, or putative indications for mode and timing of delivery.
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http://dx.doi.org/10.1038/s41467-020-18933-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552412PMC
October 2020

Forcing Seasonality of Influenza-like Epidemics with Daily Solar Resonance.

iScience 2020 Oct 24;23(10):101605. Epub 2020 Sep 24.

University of Milano, Department of Pathophysiology and Transplantation, Milano, MI 20157, Italy.

Seasonality of acute viral respiratory diseases is a well-known and yet not fully understood phenomenon. Several models have been proposed to explain the regularity of yearly recurring outbreaks and the phase differences observed at different latitudes on the Earth. Such models consider known internal causes, primarily the periodic emergence of new virus variants that evade the host immune response. Yet, this alone is generally unable to explain the regularity of recurrences and the observed phase differences. Here we show that seasonality of viral respiratory diseases, as well as its distribution with latitude on the Earth, can be fully explained by the virucidal properties of UV-B and UV-A solar photons through a daily, minute-scale, resonant forcing mechanism. Such an induced periodicity can last, virtually unperturbed, from tens to hundreds of cycles, and even in the presence of internal dynamics (host's loss of immunity) much slower than seasonal will, on a long period, generate seasonal oscillations.
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http://dx.doi.org/10.1016/j.isci.2020.101605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513765PMC
October 2020

Sterol metabolism modulates susceptibility to HIV-1 Infection.

AIDS 2020 09;34(11):1593-1602

Department of Biomedical and Clinical Sciences "L. Sacco".

Background: 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG), which catalyzes the synthesis of 25-hydroxycholesterol (25HC). 25HC intervenes in metabolic and infectious processes and controls cholesterol homeostasis and influences viral entry into host cells. We verified whether natural resistance to HIV-1 infection in HIV-1-exposed seronegative (HESN) individuals is at least partially mediated by particularities in sterol biosynthesis.

Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) isolated from 15 sexually exposed HESN and 15 healthy controls were in vitro HIV-1-infected and analyzed for: percentage of IFNα-producing plasmacytoid dendritic cells (pDCs); cholesterol signaling and inflammatory response RNA expression; resistance to HIV-1 infection. MDMs from five healthy controls were in vitro HIV-1-infected in the absence/presence of exogenously added 25HC.

Results: IFNα-producing pDCs were augmented in HESN compared with healthy controls both in unstimulated and in in vitro HIV-1-infected PBMCs (P < 0.001). An increased expression of CH25H and of a number of genes involved in cholesterol metabolism (ABCA1, ABCG1, CYP7B1, LXRα, OSBP, PPARγ, SCARB1) was observed as well; this, was associated with a reduced susceptibility to in-vitro HIV-1-infection of PBMCs and MDMs (P < 0.01). Notably, addition of 25HC to MDMs resulted in increased cholesterol efflux and augmented resistance to in-vitro HIV-1-infection.

Conclusion: Results herein show that in HESN sterol metabolism might be particularly efficient. This could be related to the activation of the IFNα pathway and results into a reduced susceptibility to in-vitro HIV-1 infection. These results suggest a possible basis for therapeutic interventions to modulate HIV-1 infection.
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http://dx.doi.org/10.1097/QAD.0000000000002591DOI Listing
September 2020

Human papillomavirus in spermatozoa is efficiently removed by washing: a suitable approach for assisted reproduction.

Reprod Biomed Online 2020 May 14;40(5):693-699. Epub 2020 Feb 14.

Department of Obstetrics and Gynaecology, Sacco Clinical Sciences Institute, University of Milan Medical School, Milan, Italy.

Research Question: Is it possible, by sperm-washing spermatozoa from clinically HPV-positive men, to obtain spermatozoa free of human papillomavirus (HPV) to be employed in assisted reproduction?

Design: This was an observational study performed on HPV-positive men. Freshly ejaculated semen was collected and readily processed by gradient separation followed by swim-up from the washed pellet. The resulting fractions were seminal plasma, cell pellet, round cells, non-motile spermatozoa and motile spermatozoa. All fractions were then tested for the presence of HPV DNA.

Results: Of the 15 clinically HPV-positive subjects, 67% were positive in at least one of the seminal fractions. If any postivity was detected, the plasma was always HPV positive. No consistent pattern was observed throughout different samples in the cell pellet, round cell and non-motile spermatozoa fractions. However, after the sperm-wash procedure, the fraction of motile spermatozoa was never found to be HPV-positive.

Conclusions: The sperm-washing technique, which was previously successfully used to remove human immunodeficiency virus, can efficiently remove HPV from spermatozoa. However, the present study was conducted on a small population so a larger follow-up study is recommended. HPV screening should be performed in sperm samples and, upon HPV positivity, sperm-washing should be considered before assisted reproduction techniques are used.
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http://dx.doi.org/10.1016/j.rbmo.2020.01.030DOI Listing
May 2020

IL-21 is associated with natural resistance to HIV-1 infection in a Colombian HIV exposed seronegative cohort.

Microbes Infect 2020 09 7;22(8):371-374. Epub 2019 Dec 7.

Department of Biomedical and Clinical Sciences 'L. Sacco', University of Milan, Milan, Italy. Electronic address:

Higher IL-21 levels were associated with natural resistance to HIV infection in an Italian cohort. Thus we wanted to confirm such association in HIV exposed seronegative individuals (HESN) from Colombia. Cells from HESN were less susceptible to infection and expressed higher IL-21 mRNA levels than healthy controls at both baseline and 7-days post-infection; similar results were observed for IL-6, perforin, and granzyme. These results suggest that IL-21/IL-6 increase may be a distinctive quality in the profile of HIV-1 resistance, at least during sexual exposure. However, further studies are necessary to confirm the specific protective mechanisms of these cytokines.
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http://dx.doi.org/10.1016/j.micinf.2019.11.002DOI Listing
September 2020

Endoplasmic Reticulum Associated Aminopeptidase 2 (ERAP2) Is Released in the Secretome of Activated MDMs and Reduces HIV-1 Infection.

Front Immunol 2019 16;10:1648. Epub 2019 Jul 16.

Department of Biomedical and Clinical Sciences L. Sacco, University of Milan, Milan, Italy.

Haplotype-specific alternative splicing of the endoplasmic reticulum (ER) aminopeptidase type 2 (ERAP2) gene results in either full-length (FL, haplotype A) or alternatively spliced (AS, haplotype B) mRNA. HapA/HapA homozygous (HomoA) subjects show a reduced susceptibility to HIV-1 infection, probably secondary to the modulation of the antigen processing/presenting machinery. ERAP1 was recently shown to be secreted from the plasma membrane in response to activation; we investigated whether ERAP2 can be released as well and if the secreted form of this enzyme retains its antiviral function. Human monocyte derived macrophages (MDMs) were differentiated from peripheral blood mononuclear cells (PBMCs) isolated from 6 HomoA healthy controls and stimulated with IFNγ and LPS. ERAP2-FL secretion was evaluated by mass spectrometry. PBMCs (14 HomoA and 16 HomoB) and CD8-depleted PBMCs (CD8PBMCs) (4 HomoA and 4 HomoB) were HIV-infected in the absence/presence of recombinant human ERAP2-FL (rhERAP2) protein; p24 viral antigen quantification was used to assess viral replication. IFNγ and CD69 mRNA expression, as well as the percentage of perforin-producing CD8+ T Lymphocytes, were analyzed 3 and 7-days post HIV-1-infection, respectively. The effect of rhERAP2 addition in cell cultures on T cell apoptosis, proliferation, activation, and maturation was evaluated as well on 24 h-stimulated PBMCs. ERAP2 can be secreted from human MDMs in response to IFNγ/LPS stimulation. Notably, the addition of rhERAP2 to PBMC and CD8PBMC cultures resulted in the reduction of viral replication, though these differences were statistically significant only in PBMCs ( < 0.05 in both HomoA and HomoB). This protective effect was associated with an increase in IFNγ and CD69 mRNA expression and in the percentage of perforin-expressing CD107CD8 cells. RhERAP2 addition also resulted in an increase in CD8 activated lymphocyte (CD25HLADRII) and Effector Memory/Terminally differentiated CD8 T cells ratio. This is the first report providing evidence for the release of ERAP2 in the secretome of immunocompetent cells. Data herein also indicate that exogenous ERAP2-FL exerts its protective function against HIV-1 infection, even in HomoB subjects who do not genetically produce it. Presumably, this defensive extracellular feature is only partially dependent on immune system modulation.
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http://dx.doi.org/10.3389/fimmu.2019.01648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646713PMC
October 2020

Evaluation of salivary and plasma microRNA expression in patients with Sjögren's syndrome, and correlations with clinical and ultrasonographic outcomes.

Clin Exp Rheumatol 2019 May-Jun;37 Suppl 118(3):70-77. Epub 2019 Jan 10.

Department of Biomedical and Clinical Sciences, Luigi Sacco University Hospital, Milan, Italy.

Objectives: To correlate the expression of microRNAs (miRNAs) 146a/b, 16, the 17-92 cluster and 181a in salivary and plasma samples taken from primary Sjögren's syndrome (pSS) patients with clinical, laboratory and ultrasound findings.

Methods: Plasma and salivary samples were collected from 28 patients with pSS according to 2012 ACR and/or 2016 ACR/EULAR criteria (27 females, mean age 64.4±10.1 years, mean disease duration 10.7±6.9 years), and from 23 healthy subjects used as controls. The following patient data were recorded: ESSDAI and ESSPRI scores, anti-SSA and anti-SSB antibody status and laboratory data, Schirmer's test, ultrasound scores of the four major salivary glands according to Cornec et al., and concomitant treatments. The retro-transcribed and quantified miRNAs were: miR16-5p, miR17-5p, miR18a-5p, miR19a-5p, miR19b-1-5p, miR20a, miR92-5p, miR146a-5p, miR146b-5p, miR181a-5p.

Results: SS patients had higher expression of salivary miR146a than gender- and age-matched controls (p=0.01). Spearman's regression analysis revealed that salivary miR146b was significantly more expressed in the patients with worse ESSPRI scores (p=0.02), whereas salivary miR17 and 146b and plasma miR17 expression was lower in the patients with higher ultrasound scores (respectively p=0.01, p=0.01 and p=0.04). Salivary miR18a expression was significantly increased in the patients who were anti-La/SSB positive (p=0.04). Neither salivary nor plasma miRNAs correlated with disease duration or concomitant therapies.

Conclusions: Our data show that salivary mi146a may represent a marker of the disease, and that the expression of salivary miR17, 18a and 146b may be altered in patients with pSS, and associated with worse ultrasound and ESSPRI scores and anti-La/SSB positivity.
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October 2019

Interleukin 21 (IL-21)/microRNA-29 (miR-29) axis is associated with natural resistance to HIV-1 infection.

AIDS 2018 11;32(17):2453-2461

Department of Biomedical and Clinical Sciences 'L. Sacco', University of Milan, Milan.

Background: Interleukin-21 (IL-21) modulates HIV-1 infection through the elicitation of different antiviral mechanisms, including Th17 lineage commitment and induction of microRNA (miR)-29, a miRNA endowed with anti-HIV activity. As miR-29 expression is significantly increased in HIV-1-exposed seronegative individuals (HESN), we investigated the role of miR-29/IL21 axis in the natural control of HIV-1 infection.

Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 15 Italian sexually exposed HESN and 15 HIV-unexposed healthy controls were in-vitro infected with an R5-tropic HIV-1Ba-L strain. Seven days post HIV-1 infection we evaluated: 1) p24 production (ELISA); 2) CD4/IL-21 and CD4/IL-17 T lymphocytes (FACS); 3) IL-17 concentration in supernatants (ELISA); and 4) IL-6, IL-17, IL-21, and miR-29a,b,c expression by CD4 T lymphocytes as well as perforin and granzyme by peripheral blood mononuclear cells (qPCR). The same analyses were performed on the 15 HIV-positive partners.

Results: At baseline IL-6 expression alone was increased in HESN compared to healthy controls. Seven days after in-vitro HIV-1 infection, nevertheless, differences emerged. Thus, CD4/IL21 and CD4/IL17 T lymphocytes, as well as IL-21 and IL-17 expression and production were significantly augmented in HESN compared to healthy controls. Interestingly, IL-21 upregulation correlated with a significantly increased expression of miR-29a,b,c and a reduced susceptibility to in-vitro HIV-1 infection in HESN alone. No differences were observed in perforin and granzyme expression.

Conclusion: The IL-21/miR-29 axis is upregulated by HIV-1 infection in HESN suggesting its involvement in the natural resistance to HIV-1 infection in HESN. Approaches that exogenously increase IL-21 production or prompt preexisting cellular IL-21 reservoir could confine the magnitude of the initial HIV-1 infection.
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http://dx.doi.org/10.1097/QAD.0000000000001938DOI Listing
November 2018

Evaluation of adhesion molecules and immune parameters in HIV-infected patients treated with an atazanavir/ritonavir- compared with a lopinavir/ritonavir-based regimen.

J Antimicrob Chemother 2018 08;73(8):2162-2170

Infectious Diseases Clinic, Azienda Socio Sanitaria Territoriale di Monza, San Gerardo Hospital-University of Milano-Bicocca, Monza, Italy.

Objectives: To evaluate changes in pro-atherosclerotic biomarkers and endothelial function in patients initiating two different PI-based regimens as part of ART.

Design: Prospective randomized 24 week study. Treatment-naive HIV-infected patients with CD4+ T cell count >250 cells/mm3 started PI-based regimens including atazanavir/ritonavir (Group A) or lopinavir/ritonavir (Group B) and were followed up in an observational follow-up study until week 96.

Methods: The expression of immune activation and adhesion molecules on CD4+ and CD8+ cells and plasma cytokine levels were assessed at weeks 0, 4, 12, 24, 48, 72 and 96. Flow-mediated dilation (FMD), pulse-wave velocity (PWV) and intima-media thickness (IMT) were measured at weeks 0 and 24. Median changes within (signed rank test) and between (Wilcoxon test) arms were calculated.

Results: Twenty-seven patients were enrolled, of whom 15 were treated with atazanavir/ritonavir and 12 with lopinavir/ritonavir. After 96 weeks of ART, CD25+/CD8+ T cells and plasma concentration of MCP-1/CCL-2 rose whereas CD44+/CD8+ T cells decreased significantly in both groups. Differences between treatments were noted for HLA-DRII+/CD8+, CD44+/CD4+ and CD11a+/CD4+, with significant increases in Group B versus Group A. No differences between groups regarding IMT, PWV and FMD were found at baseline and week 24.

Conclusions: ART initiation with PI-based regimens led to a decrease in pro-atherosclerotic biomarkers at week 24, which then rebounded at week 96. Lopinavir/ritonavir treatment resulted in an unfavourable modulation of such markers compared with atazanavir/ritonavir treatment.
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http://dx.doi.org/10.1093/jac/dky178DOI Listing
August 2018

The NLRP3 Inflammasome Is Upregulated in HIV-Infected Antiretroviral Therapy-Treated Individuals with Defective Immune Recovery.

Front Immunol 2018 12;9:214. Epub 2018 Feb 12.

Chair of Immunology, Department of Biomedical and Clinical Sciences "L. Sacco", University of Milan, Milan, Italy.

Background: Inflammasome-mediated activation of caspase-1 regulates inflammatory responses and pyroptosis. We analyzed possible associations between inflammasome-related genes and immune reconstitution in HIV-infected antiretroviral therapy (ART)-treated patients.

Methods: Cross-sectional, case-control study. HIV-infected patients on ART for ≥24 months with HIV-RNA<50 cp/mL for ≥12 months were enrolled and defined as immunological responders (IR) or non-responders (INR) if CD4 count was ≥500 or ≤350 cells/μL, respectively. Expression of inflammasome genes, caspases 1, 3, 4, 5 and γ-interferon-inducible protein 16 (IFI16) was measured in unstimulated and LPS- or aldrithiol-2-treated HIV-1 virions-stimulated peripheral blood mononuclear cells. Microbial translocation markers were evaluated.

Results: Thirty-nine patients (22 IRs; 17 INRs) were enrolled. LPS-stimulated inflammasome genes were significantly upregulated in INRs. Whereas HIV-1 stimulation induced (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) expression in both IRs and INRs, NLRP3 and IL-18 expression was significantly increased in INRs compared to IRs. Significant higher caspase-1 expression was seen as well, whereas caspase 3, 4, and 5 expression was similar in both groups. No differences in microbial translocation markers (LPS and soluble CD14) were detected in the two groups.

Conclusion: Upregulation of NLRP3 and caspase-1 is observed in INR patients. This could play a role in persistent immune activation that characterize INRs. Caspase-1 upregulation could induce CD4 T-cell loss pyroptosis, contributing to unsatisfactory CD4 T-cells recovery.
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http://dx.doi.org/10.3389/fimmu.2018.00214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816335PMC
April 2019

Humoral and cell-mediated immune responses after a booster dose of HBV vaccine in HIV-infected children, adolescents and young adults.

PLoS One 2018 14;13(2):e0192638. Epub 2018 Feb 14.

Chair of Immunology, Department of Biomedical and Clinical Sciences "L. Sacco", University of Milan, Milan, Italy.

Objective: HBV vaccine induces protective antibodies only in 23-56% of HIV-infected children. The aim of our study is to evaluate the immunologic effects of a booster dose of HBV vaccine in HIV-infected youth.

Design: 53 young HIV-infected patients in whom HBV vaccination did not elicit protective Ab titers were enrolled. All patients were on ART with optimal immunological and viral response.

Method: All patients received a booster dose of HBV vaccine (HBVAXPRO 10 μg i.m.). HBV-specific Ab titer, viral load and CD4+ T cells were measured at baseline (T0), T1, T6 and T12 months. In a subgroup of 16 patients HBV-specific cell mediated immune responses were evaluated at baseline, at T1 and T6.

Results: The booster dose induced seroconversion in 51% of patients at T1, 57% at T6, and49% at T12; seroconversion rate was significantly correlated with CD4+T cells at T0 and to the CD4 nadir. The booster dose induced HBV-specific cell mediated immunity at T6 mainly in Responders (Rs): Effector Memory CD8+T cells, HBV-specific TNFα-, IFNγ-, granzyme secreting CD8+ T cells and IL2-secreting CD4+ T cells were significantly increased in Rs compared to T0. In Non Responders (NRs), HBV-specific IL2-secreting CD4+ T cells, Central and Effector Memory CD8+ T cells were the only parameters modified at T6.

Conclusions: Seroconversion induced by a booster dose of vaccine correlates with the development of T cell immunological memory in HIV-infected patients who did not respond to the standard immunization. Alternate immunization schedules need to be considered in NRs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192638PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812642PMC
April 2018

The Immunogenicity of Branded and Biosimilar Infliximab in Rheumatoid Arthritis According to Th9-Related Responses.

Int J Mol Sci 2017 Oct 12;18(10). Epub 2017 Oct 12.

Department of Biomedical and Clinical Sciences, Azienda Ospedaliera-Polo Universitario Luigi Sacco, Milan 20157, Italy.

Our objective was to evaluate the immunogenicity of branded and biosimilar infliximab by detecting changes in T-helper-9 (Th9) percentages induced by an in vitro stimulation test.

Methods: Peripheral blood mononuclear cells collected from 55 consecutive rheumatoid arthritis (RA) outpatients (15 drug free, 20 successfully treated with branded infliximab, 20 branded infliximab inadequate responders) and 10 healthy controls were cultured, with or without 50 μg/mL of infliximab originator (Remicade) or 50 μg/mL of infliximab biosimilar (Remsima) for 18 h. Th9 lymphocytes were identified by means of flow cytometry as PU.1 and IRF4-expressing, IL-9-secreting CD4⁺ T cells. Furthermore, the markers CCR7 and CD45RA were used to distinguish naïve from memory IL-9 producer cells.

Results: Under unstimulated conditions, the drug-free RA patients had the highest percentages of Th9 lymphocytes. Following stimulation with branded infliximab, the percentages of PU.1 and IRF4-expressing Th9 cells, CCR7⁺, CD45RA (central memory) and CCR7, CD45RA (effector memory) cells significantly increased in the group of inadequate responders, but no significant variation was observed after exposure to the biosimilar of infliximab.

Conclusions: Th9 cells seem to be involved in the immune response to the epitopes of branded, but not biosimilar, infliximab, and this may depend on the recall and stimulation of both central and effector memory cells.
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http://dx.doi.org/10.3390/ijms18102127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666809PMC
October 2017

High Expression of Antiviral and Vitamin D Pathway Genes Are a Natural Characteristic of a Small Cohort of HIV-1-Exposed Seronegative Individuals.

Front Immunol 2017 13;8:136. Epub 2017 Feb 13.

Dipartimento di Scienze Biomediche e Cliniche-Luigi Sacco, Università Degli Studi di Milano , Milan , Italy.

Natural resistance to HIV-1 infection is influenced by genetics, viral-exposure, and endogenous immunomodulators such as vitamin D (VitD), being a multifactorial phenomenon that characterizes HIV-1-exposed seronegative individuals (HESNs). We compared mRNA expression of 10 antivirals, 5 immunoregulators, and 3 VitD pathway genes by qRT-PCR in cells of a small cohort of 11 HESNs, 16 healthy-controls (HCs), and 11 seropositives (SPs) at baseline, in response to calcidiol (VitD precursor) and/or aldithriol-2-(AT2)-inactivated HIV-1. In addition, the expression of TIM-3 on T and NK cells of six HCs after calcidiol and calcitriol (active VitD) treatments was evaluated by flow cytometry. Calcidiol increased the mRNA expression of (TIM-3; Th1-cells inhibitor) in HCs and HESNs. AT2-HIV-1 increased the mRNA expression of the activating VitD enzyme , of the endogenous antiviral proteins , and of immunoregulators and , but reduced the mRNA expression of VitD receptor () and antiviral peptides and in all groups. Remarkably, higher mRNA levels of , and of were found in HESNs compared to HCs either at baseline or after stimuli. Furthermore, calcitriol increases the percentage of CD4+ T cells expressing TIM-3 protein compared to EtOH controls. These results suggest that high mRNA expression of antiviral and VitD pathway genes could be genetically determined in HESNs more than viral-induced at least in peripheral blood mononuclear cells. Moreover, the virus could potentiate bio-activation and use of VitD, maintaining the homeostasis of the immune system. Interestingly, VitD-induced TIM-3 on T cells, a T cell inhibitory and anti-HIV-1 molecule, requires further studies to analyze the functional outcomes during HIV-1 infection.
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http://dx.doi.org/10.3389/fimmu.2017.00136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303892PMC
February 2017

Immune correlates of protection against HIV infection and how to elicit them.

Mucosal Immunol 2017 05 25;10(3):827-828. Epub 2017 Jan 25.

Department of Biomedical and Clinical Sciences, University of Milano, Milano, Italy.

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http://dx.doi.org/10.1038/mi.2016.134DOI Listing
May 2017

Occupational HIV Infection in a Research Laboratory With Unknown Mode of Transmission: A Case Report.

Clin Infect Dis 2017 03;64(6):810-813

Division of Infectious Diseases, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy.

A laboratory worker was infected with human immunodeficiency virus (HIV) type 1 in a biosafety level 2 containment facility, without any apparent breach. Through full-genome sequencing and phylogenetic analyses, we could identify the source of infection in a replication-competent clone that unknowingly contaminated a safe experiment. Mode of transmission remains unclear. Caution is warranted when handling HIV-derived constructs.
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http://dx.doi.org/10.1093/cid/ciw851DOI Listing
March 2017

Cell-Mediated Immunity in HIV-Infected Males With Human Papillomavirus-Related Anal Dysplastic Lesions.

Clin Infect Dis 2016 11 30;63(10):1396-1398. Epub 2016 Aug 30.

Clinic of Infectious Diseases, Department of Health Sciences, ASST Santi Paolo e Carlo.

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http://dx.doi.org/10.1093/cid/ciw590DOI Listing
November 2016

Pseudo-Mannosylated DC-SIGN Ligands as Immunomodulants.

Sci Rep 2016 10 13;6:35373. Epub 2016 Oct 13.

Department of Pathophysiology and Transplantation, University of Milan, Via F.lli VCervi 93, 20090 Milan, Italy.

DC-SIGN, a C-type lectin mainly expressed by DCs, mediates antigen uptake and can induce specific immune responses, depending on the ligand involved. Owing to these properties, DC-SIGN is an attracting target for approaches aimed at tailoring the immune response towards specific immunologic outcomes. A multivalent DC-SIGN ligand (Polyman26), containing at its core a fluorescent "rod-like" spacer and able to inhibit DC-SIGN mediated HIV infection in nanomolar concentration, has been recently developed by our group. We investigated the internalization pattern and the ability of Polyman26 to elicit innate immune responses. Results obtained by confocal microscopy indicate that Polyman26 is internalized by DCs via receptor- mediated endocytosis and is then routed to endolysosomal compartments, thus being presented together with MHC class II molecules, with important implications for the development of vaccines. Moreover, Polyman26 up-regulated the production of β-chemokines and pro-inflammatory cytokines (including IL-1β, IL-6, IL-12, and TNFα) as well as the expression of TLR9 and CD40L. These results indicate that glycomimetic DC-SIGN ligands should be further investigated and suggest that these compounds could be used to differentially stimulate immune responses.
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http://dx.doi.org/10.1038/srep35373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062166PMC
October 2016

A 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 confers protections against HIV-1 infection.

Microbes Infect 2017 Jan 18;19(1):69-74. Epub 2016 Sep 18.

Don C. Gnocchi Foundation ONLUS, IRCCS, 20148, Milan, Italy.

We investigated whether a 6-amino acid insertion/deletion polymorphism in the mucin domain of TIM-1 (T-cell immunoglobulin and mucin domain 1), modulates susceptibility to HIV-1 infection. The polymorphism was genotyped in three case/control cohorts of HIV-1 exposed seronegative individuals (HESN) and HIV-1 infected subjects from Italy, Peru, and Colombia; data from a Thai population were retrieved from the literature. Across all cohorts, homozygosity for the short TIM-1 allele was more common in HESNs than in HIV-1 infected subjects. A meta-analysis of the four association analyses yielded a p value of 0.005. In vitro infection assays of CD4+ T lymphocytes indicated that homozygosity for the short allele is associated with lower rate of HIV-1 replication. These results suggest that the deletion allele protects from HIV-1 infection with a recessive effect.
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http://dx.doi.org/10.1016/j.micinf.2016.09.005DOI Listing
January 2017

Maraviroc Reduces Arterial Stiffness in PI-Treated HIV-infected Patients.

Sci Rep 2016 06 29;6:28853. Epub 2016 Jun 29.

Chair of Immunology, Department of Biomedical and Clinical Sciences "L. Sacco", University of Milan, Milan, Italy.

The Δ32-CCR5 deletion of the CCR5 receptor is protective toward coronary artery pathology and myocardial infarction. Maraviroc (MVC), a CCR5 antagonist, was recently introduced in the therapy of HIV infection; we evaluated whether this drug could modulate the atherosclerotic burden in aviremic PI-treated HIV-positive individuals who underwent MVC intensification. Thus, the effect of MVC on intima media thickness, arterial stiffness, metabolic parameters, pro-inflammatory cytokines, endothelial dysfunction, and microbial traslocation markers was analyzed in 6 aviremic PI-treated HIV-positive individuals and were compared to those obtained in 9 additional aviremic PI-treated subjects that were enrolled retrospectively from our outpatients cohort. MVC intensification resulted in a significant reduction in intima media thickness, pulse wave velocity and triglycerides compared to baseline. Notably, MVC was also associated with a significant reduction of IL-6, microbial translocation indexes, sICAM and sVCAM; these changes were maintained throughout the 6 months of MVC intensification. No significant modifications were observed in CD4 counts, HIV viral load, and cholesterolemia. Results herein support a role of CCR5 antagonists in reducing the cardiovascular risk in HIV-infection. The hampering of inflammation, microbial translocation and the improvement of endothelial function could justify the protective role of CCR5 antagonists on atherosclerotic burden.
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http://dx.doi.org/10.1038/srep28853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926207PMC
June 2016

Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication.

Sci Rep 2016 06 2;6:27148. Epub 2016 Jun 2.

Don C Gnocchi Foundation, Milano, Italy.

Nitazoxanide (Alinia(®), NTZ) and tizoxanide (TIZ), its active circulating metabolite, belong to a class of agents known as thiazolides (TZD) endowed with broad anti-infective activities. TIZ and RM-4848, the active metabolite of RM-5038, were shown to stimulate innate immunity in vitro. Because natural resistance to HIV-1 infection in HIV-exposed seronegative (HESN) individuals is suggested to be associated with strong innate immune responses, we verified whether TIZ and RM-4848 could reduce the in vitro infectiousness of HIV-1. Peripheral blood mononuclear cells (PBMCs) from 20 healthy donors were infected in vitro with HIV-1BaL in the presence/absence of TIZ or RM4848. HIV-1 p24 were measured at different timepoints. The immunomodulatory abilities of TZD were evaluated by the expression of type I IFN pathway genes and the production of cytokines and chemokines. TZD drastically inhibited in vitro HIV-1 replication (>87%). This was associated with the activation of innate immune responses and with the up-regulation of several interferon-stimulated genes (ISGs), including those involved in cholesterol pathway, particularly the cholesterol-25 hydroxylase (CH25H). TZD inhibition of HIV-1 replication in vitro could be due to their ability to stimulate potent and multifaceted antiviral immune responses. These data warrant the exploration of TZD as preventive/therapeutic agent in HIV infection.
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http://dx.doi.org/10.1038/srep27148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890011PMC
June 2016

Identification of a Specific miRNA Profile in HIV-Exposed Seronegative Individuals.

J Acquir Immune Defic Syndr 2016 Sep;73(1):11-9

*Department of Biomedical and Clinical Sciences L. Sacco, University of Milan, Milan, Italy; †Department of Tropical Disease, University Estadual Paulista, Botucatu, Brazil; ‡Infectious Diseases Unit, S. Maria Annunziata Hospital Florence, Italy; §Department Pathophysiology and Transplantation, University of Milan, Italy; and ‖Don Gnocchi Foundation, ONLUS, IRCCS, Milan, Italy.

Objective: MicroRNAs (miRNAs) are small noncoding RNAs involved in the posttranscriptional regulation of gene expression that play important roles in viral infections. Alterations of specific miRNAs are described in HIV infection, suggesting a role for miRNAs in pathogenesis of this disease. We verified whether a particular miRNA signature could be identified in natural resistance to HIV-1.

Methods: Expression level of 84 miRNAs was analyzed by RT-qPCR in plasma and unstimulated peripheral blood mononuclear cell (PBMC) of 30 seronegative individuals repeatedly exposed to HIV-1 (HESN), 30 HIV seropositive subjects (HIV+), and 30 healthy controls (HC). Results were confirmed by individual RT-qPCR in in vitro HIV-1-infected PBMC and in their cell culture medium. Dicer and Drosha expression was analyzed in basal PBMC.

Results: Whereas Dicer and Drosha expression was comparable in HESN, HIV+ and HC, several miRNAs were upregulated both in HESN and HIV+ compared with HC. Furthermore, miRNA-29a and miR-223 were upregulated in both unstimulated PBMC and plasma of HESN alone; their expression was reduced upon in vitro HIV-1 infection of HESN PBMC indicating that, upon infection, they are secreted in the extracellular milieu. These results were confirmed by individual qPCR.

Conclusions: Our studies demonstrate that HIV-1 exposure modifies miRNAs expression even in the absence of productive infection. Because those miRNAs that are specifically increased only in HESN have been known to reduce HIV-1 replication, their modulation could represent an important mechanism in resistance to HIV-1 infection.
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http://dx.doi.org/10.1097/QAI.0000000000001070DOI Listing
September 2016

Multiple inflammasome complexes are activated in autistic spectrum disorders.

Brain Behav Immun 2016 Oct 12;57:125-133. Epub 2016 Mar 12.

Don C. Gnocchi Foundation, IRCCS, Piazza Morandi, 3, 20121 Milano, Italy; Department of Physiopathology and Transplants, University of Milano, Milano, Italy.

Background: Inflammasomes are multimeric protein platforms involved in the regulation of inflammatory responses whose activity results in the production of proinflammatory cytokines. Because neuroinflammation is observed in autistic spectrum disorders (ASD), a neurologic condition of childhood resulting in a complex behavioural impairment, we analyzed the inflammasomes activity in ASD. Additionally we verified whether alterations of the gastrointestinal (GI) barriers might play a role in inflammasomes activation.

Methods: The activity of the inflammasomes, the concentration of the inflammasomes-derived proinflammatory cytokines interleukin (IL)-1β and IL-18, and serum parameters of GI damage were analyzed in 25 ASD children, 23 healthy siblings (HS) and 30 unrelated age-matched healthy controls (HC).

Results: A significant upregulation of the AIM2 and the NLRP3 inflammasomes and an increased production of IL-1β and IL-18 that was associated with a consistent reduction of IL-33, an anti inflammation cytokine were observed in ASD alone. Notably, in a possible immune-mediated attempt to dampen inflammation, IL-37, a suppressor of innate inflammatory responses, was significantly augmented in these same children. Finally, intestinal fatty acid binding protein (IFABP), an index of altered GI permeability, was significantly increased in serum of ASD and HS.

Conclusions: These results show that the inflammasomes are activated in ASD and shed light on the molecular mechanisms responsible for ASD-associated neuroinflammation. The observation that GI alterations could be present as well in ASD offers a possible link between such alterations and neuroinflammation. Therapeutic strategies targeting inflammasome activation could be useful in ASD.
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http://dx.doi.org/10.1016/j.bbi.2016.03.009DOI Listing
October 2016