Publications by authors named "Chiara Agrati"

132 Publications

Pleural Mesothelial Cells Modulate the Inflammatory/Profibrotic Response During SARS-CoV-2 Infection.

Front Mol Biosci 2021 26;8:752616. Epub 2021 Nov 26.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely understood. In particular, no direct evidence of pleura implication in COVID-19-related fibrotic damage has been reported so far. In this study, the expression of epithelial cytokeratins and Wilms tumor 1 (WT1), specific markers of mesothelial cells (MCs), was analyzed in COVID-19 and unrelated pleura autoptic samples. SARS-CoV-2 replication was analyzed by RT-PCR and confocal microscopy in MeT5A, a pleura MC line. SARS-CoV-2 receptors were analyzed by RT-PCR and western blot. Inflammatory cytokines from the supernatants of SARS-CoV-2-infected MeT5A cells were analysed by Luminex and ELLA assays. Immunohistochemistry of COVID-19 pleura patients highlighted disruption of pleura monolayer and fibrosis of the sub-mesothelial stroma, with the presence of MCs with fibroblastoid morphology in the sub-mesothelial stroma, but no evidence of direct infection . Interestingly, we found evidence of ACE2 expression in MCs from pleura of COVID-19 patients. analysis shown that MeT5A cells expressed ACE2, TMPRSS2, ADAM17 and NRP1, plasma membrane receptors implicated in SARS-CoV-2 cell entry and infectivity. Moreover, MeT5A cells sustained SARS-CoV-2 replication and productive infection. Infected MeT5A cells produced interferons, inflammatory cytokines and metalloproteases. Overall, our data highlight the potential role of pleura MCs as promoters of the fibrotic reaction and regulators of the immune response upon SARS-CoV-2 infection.
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http://dx.doi.org/10.3389/fmolb.2021.752616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8662383PMC
November 2021

Multi-omics approach to COVID-19: a domain-based literature review.

J Transl Med 2021 12 7;19(1):501. Epub 2021 Dec 7.

National Institute for Infectious Diseases, "Lazzaro Spallanzani" - IRCCS, Via Portuense, 292, 00149, Rome, Italy.

Background: Omics data, driven by rapid advances in laboratory techniques, have been generated very quickly during the COVID-19 pandemic. Our aim is to use omics data to highlight the involvement of specific pathways, as well as that of cell types and organs, in the pathophysiology of COVID-19, and to highlight their links with clinical phenotypes of SARS-CoV-2 infection.

Methods: The analysis was based on the domain model, where for domain it is intended a conceptual repository, useful to summarize multiple biological pathways involved at different levels. The relevant domains considered in the analysis were: virus, pathways and phenotypes. An interdisciplinary expert working group was defined for each domain, to carry out an independent literature scoping review.

Results: The analysis revealed that dysregulated pathways of innate immune responses, (i.e., complement activation, inflammatory responses, neutrophil activation and degranulation, platelet degranulation) can affect COVID-19 progression and outcomes. These results are consistent with several clinical studies.

Conclusions: Multi-omics approach may help to further investigate unknown aspects of the disease. However, the disease mechanisms are too complex to be explained by a single molecular signature and it is necessary to consider an integrated approach to identify hallmarks of severity.
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http://dx.doi.org/10.1186/s12967-021-03168-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8649311PMC
December 2021

Humoral- and T-Cell-Specific Immune Responses to SARS-CoV-2 mRNA Vaccination in Patients With MS Using Different Disease-Modifying Therapies.

Neurology 2021 Nov 22. Epub 2021 Nov 22.

Clinical Division of Infectious Diseases, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy.

Objective: To evaluate the immune-specific response after the full SARS-CoV-2 vaccination of multiple sclerosis (MS) patients treated with different Disease Modifying drugs by the detection of both serological- and T-cell responses.

Methods: Health care workers (HCWs) and MS patients, having completed the two-dose schedule of an mRNA-based vaccine against SARS-CoV-2 in the last 2-4 weeks, were enrolled from two parallel prospective studies conducted in Rome, Italy, at the National Institute for Infectious diseases Spallanzani-IRCSS and San Camillo Forlanini Hospital. Serological response was evaluated by quantifying the Region-Binding-Domain (RBD) and neutralizing-antibodies. Cell-mediated response was analyzed by a whole-blood test quantifying interferon (IFN)-γ response to spike peptides. Cells responding to spike stimulation were identified by FACS analysis.

Results: We prospectively enrolled 186 vaccinated individuals: 78 HCWs and 108 MS patients. Twenty-eight MS patients were treated with IFN-β, 35 with fingolimod, 20 with cladribine, and 25 with ocrelizumab. A lower anti-RBD-antibody response rate was found in patients treated with ocrelizumab (40%, p<0.0001) and fingolimod (85.7%, p=0.0023) compared to HCWs and patients treated with cladribine or IFN-β. Anti-RBD-antibody median titer was lower in patients treated with ocrelizumab (p<0.0001), fingolimod (p<0.0001) and cladribine (p=0.010) compared to HCWs and IFN-β-treated patients. Importantly, serum neutralizing activity was present in all the HCWs tested and only in a minority of the fingolimod-treated patients (16.6%). T-cell-specific response was detected in the majority of MS patients (62%), albeit with significantly lower IFN-γ levels compared to HCWs. The lowest frequency of T-cell response was found in fingolimod-treated patients (14.3%). T-cell-specific response correlated with lymphocyte count and anti-RBD antibody titer (rho=0.554, p<0.0001 and rho=0.255, p=0.0078 respectively). Finally, IFN-γ T-cell response was mediated by both CD4 and CD8 T cells.

Conclusion: mRNA vaccines induce both humoral and cell-mediated specific immune responses against spike peptides in all HCWs and in the majority of MS patients. These results carry relevant implications for managing vaccinations suggesting to promote vaccination in all treated MS patients.

Classification Of Evidence: This study provides Class III data that COVID mRNA vaccination induces both humoral and cell-mediated specific immune responses against viral spike proteins in a majority of MS patients.
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http://dx.doi.org/10.1212/WNL.0000000000013108DOI Listing
November 2021

Predicting respiratory failure in patients infected by SARS-CoV-2 by admission sex-specific biomarkers.

Biol Sex Differ 2021 11 22;12(1):63. Epub 2021 Nov 22.

Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Background: Several biomarkers have been identified to predict the outcome of COVID-19 severity, but few data are available regarding sex differences in their predictive role. Aim of this study was to identify sex-specific biomarkers of severity and progression of acute respiratory distress syndrome (ARDS) in COVID-19.

Methods: Plasma levels of sex hormones (testosterone and 17β-estradiol), sex-hormone dependent circulating molecules (ACE2 and Angiotensin1-7) and other known biomarkers for COVID-19 severity were measured in male and female COVID-19 patients at admission to hospital. The association of plasma biomarker levels with ARDS severity at admission and with the occurrence of respiratory deterioration during hospitalization was analysed in aggregated and sex disaggregated form.

Results: Our data show that some biomarkers could be predictive both for males and female patients and others only for one sex. Angiotensin1-7 plasma levels and neutrophil count predicted the outcome of ARDS only in females, whereas testosterone plasma levels and lymphocytes counts only in males.

Conclusions: Sex is a biological variable affecting the choice of the correct biomarker that might predict worsening of COVID-19 to severe respiratory failure. The definition of sex specific biomarkers can be useful to alert patients to be safely discharged versus those who need respiratory monitoring.
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http://dx.doi.org/10.1186/s13293-021-00407-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8607227PMC
November 2021

Strong immunogenicity of heterologous prime-boost immunizations with the experimental vaccine GRAd-COV2 and BNT162b2 or ChAdOx1-nCOV19.

NPJ Vaccines 2021 Nov 4;6(1):131. Epub 2021 Nov 4.

ReiThera Srl, Rome, Italy.

Here we report on the humoral and cellular immune response in eight volunteers who autonomously chose to adhere to the Italian national COVID-19 vaccination campaign more than 3 months after receiving a single-administration GRAd-COV2 vaccine candidate in the context of the phase-1 clinical trial. We observed a clear boost of both binding/neutralizing antibodies as well as T-cell responses upon receipt of the heterologous BNT162b2 or ChAdOx1-nCOV19 vaccines. These results, despite the limitation of the small sample size, support the concept that a single dose of an adenoviral vaccine may represent an ideal tool to effectively prime a balanced immune response, which can be boosted to high levels by a single dose of a different vaccine platform.
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http://dx.doi.org/10.1038/s41541-021-00394-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8569156PMC
November 2021

Coordinated cellular and humoral immune responses after two-dose SARS-CoV2 mRNA vaccination in liver transplant recipients.

Liver Int 2022 Jan 17;42(1):180-186. Epub 2021 Nov 17.

National Institute for Infectious Diseases L. Spallanzani - IRCCS (INMI) Rome.

Limited data are available on risks and benefits of anti-SARS-CoV2 vaccination in solid organ transplant recipients, and weaker responses have been described. At the Italian National Institute for Infectious Diseases, 61 liver transplant recipients underwent testing to describe the dynamics of humoral and cell-mediated immune response after two doses of anti-SARS-CoV2 mRNA vaccines and compared with 51 healthy controls. Humoral response was measured by quantifying both anti-spike and neutralizing antibodies; cell-mediated response was measured by PBMC proliferation assay with IFN-γ and IL-2 production. Liver transplant recipients showed lower response rates compared with controls in both humoral and cellular arms; shorter time since transplantation and multi-drug immunosuppressive regimen containing mycophenolate mofetil were predictive of reduced response to vaccination. Specific antibody and cytokine production, though reduced, were highly correlated in transplant recipients.
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http://dx.doi.org/10.1111/liv.15089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8662049PMC
January 2022

Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA.

Cells 2021 09 26;10(10). Epub 2021 Sep 26.

Neonatal Intensive Care Unit and Human Milk Bank, Department of Neonatology, Bambino Gesù Children's Hospital, IRCSS, Piazza Sant'Onofrio, 4, 00165 Rome, Italy.

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA.
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http://dx.doi.org/10.3390/cells10102541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8533837PMC
September 2021

Vitamin D as Modulator of Drug Concentrations: A Study on Two Italian Cohorts of People Living with HIV Administered with Efavirenz.

Nutrients 2021 Oct 12;13(10). Epub 2021 Oct 12.

Laboratory of Clinical Pharmacology and Pharmacogenetics, Department of Medical Sciences, Amedeo di Savoia Hospital, University of Turin, 10149 Turin, Italy.

To date, vitamin D seems to have a significant role in affecting the prevention and immunomodulation in COVID-19 disease. Nevertheless, it is important to highlight that this pro-hormone has other several activities, such as affecting drug concentrations, since it regulates the expression of cytochrome P450 (CYP) genes. Efavirenz (EFV) pharmacokinetics is influenced by CYPs, but no data are available in the literature concerning the association among vitamin D levels, seasonality (which affects vitamin D concentrations) and EFV plasma levels. For this reason, the aim of this study was to evaluate the effect of 25-hydroxy vitamin D (25(OH)D3) levels on EFV plasma concentrations in different seasons. We quantified 25(OH)D3 by using chemiluminescence immunoassay, whereas EFV plasma concentrations were quantified with the HPLC-PDA method. A total of 316 patients were enrolled in Turin and Rome. Overall, 25(OH)D3levels resulted in being inversely correlated with EFV concentrations. Some patients with EFV levels higher than 4000 ng/mL showed a deficient 25(OH)D3 concentration in Turin and Rome cohorts and together. EFV concentrations were different in patients without vitamin D supplementation, whereas, for vitamin D-administered individuals, no difference in EFV exposure was present. Concerning seasonality, EFV concentrations were associated with 25(OH)D3 deficiency only in winter and in spring, whereas a significant influence was highlighted for 25(OH)D3 stratification for deficient, insufficient and sufficient values in winter, spring and summer. A strong and inverse association between 25(OH)D3and EFV plasma concentrations was suggested. These data suggest that vitamin D is able to affect drug exposure in different seasons; thus, the achievement of the clinical outcome could be improved by also considering this pro-hormone.
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http://dx.doi.org/10.3390/nu13103571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8538640PMC
October 2021

T-cell immune response after mRNA SARS-CoV-2 vaccines is frequently detected also in the absence of seroconversion in patients with lymphoid malignancies.

Br J Haematol 2021 Oct 14. Epub 2021 Oct 14.

School of Medicine, University of Milano, Italy.

Patients affected by lymphoid malignancies (LM) are frequently immune-compromised, suffering increased mortality from COVID-19. This prospective study evaluated serological and T-cell responses after complete mRNA vaccination in 263 patients affected by chronic lymphocytic leukaemia, B- and T-cell lymphomas and multiple myeloma. Results were compared with those of 167 healthy subjects matched for age and sex. Overall, patient seroconversion rate was 64·6%: serological response was lower in those receiving anti-cancer treatments in the 12 months before vaccination: 55% vs 81·9% (P < 0·001). Anti-CD20 antibody plus chemotherapy treatment was associated with the lowest seroconversion rate: 17·6% vs. 71·2% (P < 0·001). In the multivariate analysis conducted in the subgroup of patients on active treatment, independent predictors for seroconversion were: anti-CD20 treatment (P < 0·001), aggressive B-cell lymphoma diagnosis (P = 0·002), and immunoglobulin M levels <40 mg/dl (P = 0·030). The T-cell response was evaluated in 99 patients and detected in 85 of them (86%). Of note, 74% of seronegative patients had a T-cell response, but both cellular and humoral responses were absent in 13·1% of cases. Our findings raise some concerns about the protection that patients with LM, particularly those receiving anti-CD20 antibodies, may gain from vaccination. These patients should strictly maintain all the protective measures.
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http://dx.doi.org/10.1111/bjh.17877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8653177PMC
October 2021

Association of Sofosbuvir and Daclatasvir Plasma Trough Concentrations with Patient-, Treatment-, and Disease-Related Factors Among HIV/HCV-Coinfected Persons.

Eur J Drug Metab Pharmacokinet 2022 Jan 8;47(1):135-142. Epub 2021 Oct 8.

National Institute for Infectious Diseases "L. Spallanzani" I.R.C.C.S., Via Portuense 292, 00149, Rome, Italy.

Background: Sofosbuvir plus daclatasvir achieves high rates of sustained virologic response (SVR), with no differences according to HIV serostatus. However, only limited information is available on the pharmacokinetic variability of sofosbuvir and daclatasvir in HIV/HCV-coinfected patients.

Objectives: The aim of this study was to identify patient-, treatment-, and disease-related factors that are significantly associated with sofosbuvir and daclatasvir plasma trough concentrations (C), including liver and renal function, among HIV/HCV-coinfected persons.

Methods: In this observational cohort pilot study, HIV/HCV-coinfected patients undergoing sofosbuvir plus daclatasvir treatment were prospectively enrolled. Biochemical and viro-immunological parameters were assessed at baseline, week 4 (W4), end of treatment (EOT), and after EOT. The FIB-4 score and CKD-EPI equation were used to estimate liver disease and glomerular filtration rate (eGFR), respectively. For sofosbuvir, sofosbuvir metabolite (GS-331007), and daclatasvir, C was measured at W4 and week 8 (W8), and the mean of the values at those two time points (mean-C) was calculated. The Mann-Whitney test and Spearman's rank correlation were used to evaluate the correlations between the mean-C of each direct-acting antiviral (DAA) and the considered variables.

Results: Thirty-five patients were included (SVR 94%). An increased GS-331007 mean-C was significantly correlated with a decreased eGFR at W4 (rho = -0.36; p = 0.037) and EOT (rho = -0.34; p = 0.048). There was a significant correlation between daclatasvir mean-C and FIB-4 at all time points: baseline (rho = -0.35; p = 0.037), W4 (rho = -0.44; p = 0.008), EOT (rho = -0.40; p = 0.023), and after EOT (rho = -0.39; p = 0.028).

Conclusions: In HIV/HCV-coinfected patients in a real-world setting, exposure to a high GS-331007 C was associated with a slight decrease in renal function, while advanced hepatic impairment was significantly associated with a lower daclatasvir C. Though the clinical and therapeutic relevance of these findings may be limited, increasing clinicians' knowledge regarding DAA exposure in difficult-to-treat patients could be relevant in single cases, and further investigations are warranted.
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http://dx.doi.org/10.1007/s13318-021-00725-wDOI Listing
January 2022

ImmunosuppressiveTherapies Differently Modulate Humoral- and T-Cell-Specific Responses to COVID-19 mRNA Vaccine in Rheumatoid Arthritis Patients.

Front Immunol 2021 14;12:740249. Epub 2021 Sep 14.

Translational Research Unit, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy.

Objective: To assess in rheumatoid arthritis (RA) patients, treated with different immunosuppressive therapies, the induction of SARS-CoV-2-specific immune response after vaccination in terms of anti-region-binding-domain (RBD)-antibody- and T-cell-specific responses against spike, and the vaccine safety in terms of clinical impact on disease activity.

Methods: Health care workers (HCWs) and RA patients, having completed the BNT162b2-mRNA vaccination in the last 2 weeks, were enrolled. Serological response was evaluated by quantifying anti-RBD antibodies, while the cell-mediated response was evaluated by a whole-blood test quantifying the interferon (IFN)-γ-response to spike peptides. FACS analysis was performed to identify the cells responding to spike stimulation. RA disease activity was evaluated by clinical examination through the DAS28crp, and local and/or systemic clinical adverse events were registered. In RA patients, the ongoing therapeutic regimen was modified during the vaccination period according to the American College of Rheumatology indications.

Results: We prospectively enrolled 167 HCWs and 35 RA patients. Anti-RBD-antibodies were detected in almost all patients (34/35, 97%), although the titer was significantly reduced in patients under CTLA-4-inhibitors (median: 465 BAU/mL, IQR: 103-1189, p<0.001) or IL-6-inhibitors (median: 492 BAU/mL, IQR: 161-1007, p<0.001) compared to HCWs (median: 2351 BAU/mL, IQR: 1389-3748). T-cell-specific response scored positive in most of RA patients [24/35, (69%)] with significantly lower IFN-γ levels in patients under biological therapy such as IL-6-inhibitors (median: 33.2 pg/mL, IQR: 6.1-73.9, p<0.001), CTLA-4-inhibitors (median: 10.9 pg/mL, IQR: 3.7-36.7, p<0.001), and TNF-α-inhibitors (median: 89.6 pg/mL, IQR: 17.8-224, p=0.002) compared to HCWs (median: 343 pg/mL, IQR: 188-756). A significant correlation between the anti-RBD-antibody titer and spike-IFN-γ-specific T-cell response was found in RA patients (rho=0.432, p=0.009). IFN-γ T-cell response was mediated by CD4 and CD8 T cells. Finally, no significant increase in disease activity was found in RA patients following vaccination.

Conclusion: This study showed for the first time that antibody-specific and whole-blood spike-specific T-cell responses induced by the COVID-19 mRNA-vaccine were present in the majority of RA patients, who underwent a strategy of temporary suspension of immunosuppressive treatment during vaccine administration. However, the magnitude of specific responses was dependent on the immunosuppressive therapy administered. In RA patients, BNT162b2 vaccine was safe and disease activity remained stable.
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http://dx.doi.org/10.3389/fimmu.2021.740249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477040PMC
October 2021

Pharmacokinetics of bictegravir, emtricitabine and tenofovir alafenamide in a gastrectomized patient with HIV.

J Antimicrob Chemother 2021 11;76(12):3320-3322

National Institute for Infectious Diseases 'Lazzaro Spallanzani' IRCCS, Via Portuense 292, 00149 Rome, Italy.

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http://dx.doi.org/10.1093/jac/dkab319DOI Listing
November 2021

COVID-19 Vaccination in Fragile Patients: Current Evidence and an Harmonized Transdisease Trial.

Front Immunol 2021;12:704110. Epub 2021 Aug 10.

Department of Biomedical Sciences, Humanitas University, Milan, Italy.

Patients diagnosed with malignancy, neurological and immunological disorders, , fragile patients, have been excluded from COVID-19 vaccine trials. However, this population may present immune response abnormalities, and relative reduced vaccine responsiveness. Here we review the limited current evidence on the immune responses to vaccination of patients with different underlying diseases. To address open questions we present the VAX4FRAIL study aimed at assessing immune responses to vaccination in a large transdisease cohort of patients with cancer, neurological and rheumatological diseases.
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http://dx.doi.org/10.3389/fimmu.2021.704110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8383886PMC
September 2021

Expansion of Myeloid Derived Suppressor Cells Contributes to Platelet Activation by L-Arginine Deprivation during SARS-CoV-2 Infection.

Cells 2021 08 17;10(8). Epub 2021 Aug 17.

Department of Epidemiology, Pre-Clinical Research and Advanced Diagnostic, National Institute for Infectious Diseases "Lazzaro Spallanzani"-IRCCS, Via Portuense, 292-00149 Rome, Italy.

Massive platelet activation and thrombotic events characterize severe COVID-19, highlighting their critical role in SARS-CoV-2-induced immunopathology. Since there is a well-described expansion of myeloid-derived suppressor cells (MDSC) in severe COVID-19, we evaluated their possible role in platelet activation during SARS-CoV-2 infection. During COVID-19, a lower plasmatic L-arginine level was observed compared to healthy donors, which correlated with MDSC frequency. Additionally, activated GPIIb/IIIa complex (PAC-1) expression was higher on platelets from severe COVID-19 patients compared to healthy controls and inversely correlated with L-arginine plasmatic concentration. Notably, MDSC were able to induce PAC-1 expression in vitro by reducing L-arginine concentration, indicating a direct role of PMN-MDSC in platelet activation. Accordingly, we found a positive correlation between ex vivo platelet PAC-1 expression and PMN-MDSC frequency. Overall, our data demonstrate the involvement of PMN-MDSC in triggering platelet activation during COVID-19, highlighting a novel role of MDSC in driving COVID-19 pathogenesis.
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http://dx.doi.org/10.3390/cells10082111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391818PMC
August 2021

The Role of P-Selectin in COVID-19 Coagulopathy: An Updated Review.

Int J Mol Sci 2021 Jul 26;22(15). Epub 2021 Jul 26.

National Institute for Infectious Diseases "Lazzaro Spallanzani" I.R.C.C.S., Via Portuense 292, 00146 Rome, Italy.

In severe COVID-19, which is characterized by blood clots and neutrophil-platelet aggregates in the circulating blood and different tissues, an increased incidence of cardiovascular complications and venous thrombotic events has been reported. The inflammatory storm that characterizes severe infections may act as a driver capable of profoundly disrupting the complex interplay between platelets, endothelium, and leukocytes, thus contributing to the definition of COVID-19-associated coagulopathy. In this frame, P-selectin represents a key molecule expressed on endothelial cells and on activated platelets, and contributes to endothelial activation, leucocyte recruitment, rolling, and tissue migration. Briefly, we describe the current state of knowledge about P-selectin involvement in COVID-19 pathogenesis, its possible use as a severity marker and as a target for host-directed therapeutic intervention.
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http://dx.doi.org/10.3390/ijms22157942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348106PMC
July 2021

Fibrogenic signals persist in DAA-treated HCV patients after sustained virological response.

J Hepatol 2021 12 13;75(6):1301-1311. Epub 2021 Jul 13.

National Institute for Infectious Diseases L. Spallanzani, IRCCS, Italy; Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. Electronic address:

Background & Aims: Patients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary.

Methods: Extracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot.

Results: On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p <0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (Log fold change of 4.4).

Conclusions: Taken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR.

Lay Summary: Direct-acting antivirals lead to virological cure in the majority of patients with chronic hepatitis C virus infection. However, the risk of liver disease progression or complications in patients with fibrosis and cirrhosis remains in some patients even after virological cure. Herein, we show that extracellular vesicle modifications could be linked to long-term liver disease progression in patients who have achieved virological cure; these modifications could potentially be used as biomarkers or treatment targets in such patients.
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http://dx.doi.org/10.1016/j.jhep.2021.07.003DOI Listing
December 2021

Coordinate Induction of Humoral and Spike Specific T-Cell Response in a Cohort of Italian Health Care Workers Receiving BNT162b2 mRNA Vaccine.

Microorganisms 2021 Jun 16;9(6). Epub 2021 Jun 16.

National Institute for Infectious Diseases (INMI) L. Spallanzani-IRCCS, Via Portuense 292, 00149 Roma, Italy.

Vaccination is the main public health measure to reduce SARS-CoV-2 transmission and hospitalization, and a massive worldwide scientific effort resulted in the rapid development of effective vaccines. This work aimed to define the dynamics of humoral and cell-mediated immune response in a cohort of health care workers (HCWs) who received a two-dose BNT162b2-mRNA vaccination. The serological response was evaluated by quantifying the anti-RBD and neutralizing antibodies. The cell-mediated response was performed by a whole blood test quantifying Th1 cytokines (IFN-γ, TNF-α, IL-2), produced in response to spike peptides. The BNT162b2-mRNA vaccine induced both humoral and cell-mediated immune responses against spike peptides in virtually all HCWs without previous SARS-CoV-2 infection, with a moderate inverse relation with age in the anti-RBD response. Spike-specific T cells produced several Th1 cytokines (IFN-γ, TNF-α, and IL-2), which correlated with the specific-serological response. Overall, our study describes the ability of the BNT162b2 mRNA vaccine to elicit a coordinated neutralizing humoral and spike-specific T cell response in HCWs. Assessing the dynamics of these parameters by an easy immune monitoring protocol can allow for the evaluation of the persistence of the vaccine response in order to define the optimal vaccination strategy.
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http://dx.doi.org/10.3390/microorganisms9061315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235087PMC
June 2021

Impact of Prior Influenza and Pneumoccocal Vaccines on Humoral and Cellular Response to SARS-CoV-2 BNT162b2 Vaccination.

Vaccines (Basel) 2021 Jun 8;9(6). Epub 2021 Jun 8.

National Institute for Infectious Diseases Lazzaro Spallanzani, 00149 Rome, Italy.

Vaccination against SARS-CoV-2 is considered the most effective method of prevention to contain the pandemic. While highly effective SARS-CoV-2 vaccines are being applied on a large-scale, whether and to what extent the strength of the vaccine-induced immune response could be further potentiated is still an object of debate. Several reports studied the effect of different vaccines on the susceptibility and mortality of COVID-19, with conflicting results. We aimed to evaluate whether previous influenza and/or pneumococcal vaccination had an impact on the specific immune response to the SARS-CoV-2 BNT162b2 mRNA vaccine. The study population consists of 710 workers from our Institute who completed the BNT162b2 schedule and have been tested at least once after the second dose, from 27 December 2020 up to 15 April 2021. Of these, 152 (21.4%) had received an influenza and 215 (30.3%) a concomitant influenza and pneumococcal vaccination, a median of 102 days before the second dose of BNT162b2. Overall, 100% of workers were tested for anti-Spike receptor-binding domain (anti-S/RBD) antibodies, 224 workers for neutralization titer (Micro-neutralization assay, MNA), and 155 workers for a spike-specific T cell interferon-γ response (IFN-γ). The levels of anti-S/RBD, MNA and IFN-γ were evaluated and compared according to sex, age, involvement in direct care of COVID-19 patients, and previous influenza/pneumococcal vaccination. At the univariate analysis, no statistically significant association was observed with regard to a previous influenza and pneumococcal vaccination. A significant lower anti-S/RBD response was observed according to an older age and male sex, while MNA titers were significantly associated to sex but not to age. At the multivariable analysis, workers receiving a concomitant influenza and pneumococcal vaccination or only influenza showed a 58% ( 0.01) and 42% ( 0.07) increase in MNA titers, respectively, compared to those who did not receive an influenza/pneumococcal vaccination. Female workers showed an 81% MNA and a 44% anti-S/RBD increase compared to male workers ( < 0.001). Compared to workers aged 21 to 49 years, those aged 50 or older were associated with a reduction in the anti-S/RBD (16%; 0.005), MNA (31%; 0.019), and IFN.g (32%) immune response. Maintaining the influenza and pneumococcal immunization program for the coming season, in which COVID-19 could still be spreading, remains strongly recommended to protect those who are more vulnerable and to limit the potential burden of these infections on the healthcare system.
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http://dx.doi.org/10.3390/vaccines9060615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229609PMC
June 2021

High Levels of TRIM5α Are Associated with Xenophagy in HIV-1-Infected Long-Term Nonprogressors.

Cells 2021 05 14;10(5). Epub 2021 May 14.

Department of Epidemiology, Preclinical Research and Advanced Diagnostics, National Institute for Infectious Diseases, Lazzaro Spallanzani-IRCCS, 00149 Rome, Italy.

Autophagy is a lysosomal-dependent degradative mechanism essential in maintaining cellular homeostasis, but it is also considered an ancient form of innate eukaryotic fighting against invading microorganisms. Mounting evidence has shown that HIV-1 is a critical target of autophagy that plays a role in HIV-1 replication and disease progression. In a special subset of HIV-1-infected patients that spontaneously and durably maintain extremely low viral replication, namely, long-term nonprogressors (LTNP), the resistance to HIV-1-induced pathogenesis is accompanied, in vivo, by a significant increase in the autophagic activity in peripheral blood mononuclear cells. Recently, a new player in the battle of autophagy against HIV-1 has been identified, namely, tripartite motif protein 5α (TRIM5α). In vitro data demonstrated that TRIM5α directly recognizes HIV-1 and targets it for autophagic destruction, thus protecting cells against HIV-1 infection. In this paper, we analyzed the involvement of this factor in the control of HIV-1 infection through autophagy, in vivo, in LTNP. The results obtained showed significantly higher levels of TRIM5α expression in cells from LTNP with respect to HIV-1-infected normal progressor patients. Interestingly, the colocalization of TRIM5α and HIV-1 proteins in autophagic vacuoles in LTNP cells suggested the participation of TRIM5α in the autophagy containment of HIV-1 in LTNP. Altogether, our results point to a protective role of TRIM5α in the successful control of the chronic viral infection in HIV-1-controllers through the autophagy mechanism. In our opinion, these findings could be relevant in fighting against HIV-1 disease, because autophagy inducers might be employed in combination with antiretroviral drugs.
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http://dx.doi.org/10.3390/cells10051207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156091PMC
May 2021

Prophylactic heparin and risk of orotracheal intubation or death in patients with mild or moderate COVID-19 pneumonia.

Sci Rep 2021 05 31;11(1):11334. Epub 2021 May 31.

Centre for Clinical Research, Epidemiology, Modelling and Evaluation (CREME), Institute for Global Health, UCL, London, UK.

Prophylactic low molecular weight heparin (pLMWH) is currently recommended in COVID-19 to reduce the risk of coagulopathy. The aim of this study was to evaluate whether the antinflammatory effects of pLMWH could translate in lower rate of clinical progression in patients with COVID-19 pneumonia. Patients admitted to a COVID-hospital in Rome with SARS-CoV-2 infection and mild/moderate pneumonia were retrospectively evaluated. The primary endpoint was the time from hospital admission to orotracheal intubation/death (OTI/death). A total of 449 patients were included: 39% female, median age 63 (IQR, 50-77) years. The estimated probability of OTI/death for patients receiving pLMWH was: 9.5% (95% CI 3.2-26.4) by day 20 in those not receiving pLMWH vs. 10.4% (6.7-15.9) in those exposed to pLMWH; p-value = 0.144. This risk associated with the use of pLMWH appeared to vary by PaO/FiO ratio: aHR 1.40 (95% CI 0.51-3.79) for patients with an admission PaO/FiO ≤ 300 mmHg and 0.27 (0.03-2.18) for those with PaO/FiO > 300 mmHg; p-value at interaction test 0.16. pLMWH does not seem to reduce the risk of OTI/death mild/moderate COVID-19 pneumonia, especially when respiratory function had already significantly deteriorated. Data from clinical trials comparing the effect of prophylactic vs. therapeutic dosage of LMWH at various stages of COVID-19 disease are needed.
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http://dx.doi.org/10.1038/s41598-021-90713-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167179PMC
May 2021

Synergy Between Vitamin D and Sex Hormones in Respiratory Functionality of Patients Affected by COVID-19.

Front Pharmacol 2021 13;12:683529. Epub 2021 May 13.

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

The outcome of COVID-19 appears to be influenced by vitamin D status of population. Although epidemiological data indicate that COVID-19 produces more severe symptoms and higher mortality in elderly in comparison to young patients and in men in comparison to women to date sex and age differences in vitamin D status in infected patients have not been evaluated yet. In this study we evaluated the levels of circulating 25(OH)D in patients hospitalized for COVID-19 divided accordingly to their sex and age. We also correlated 25(OH)D levels with patient's respiratory status (i.e., PaO2/FiO2 ratio) and with sex hormones plasma levels to analyze the potential relationship of these parameters. We found no significant differences in plasma levels of 25(OH)D between pre- and post-menopausal female patients and age matched male patients. Interestingly, the 25(OH)D plasma levels positively correlated to PaO2/FiO2 ratio only in young patients, regardless of their sex. We also found a significantly positive correlation between 17β-estradiol and 25(OH)D in elderly women and between testosterone and 25(OH)D in elderly men, supporting the role of sex hormones in maintaining 25(OH)D levels. In conclusion, we suggest that a synergy between vitamin D and sex hormones could contribute to the age-related outcome of COVID-19.
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http://dx.doi.org/10.3389/fphar.2021.683529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155348PMC
May 2021

Plasma concentrations of remdesivir metabolite in a critical COVID-19 patient needing continuous venovenous haemodialysis.

Eur J Clin Pharmacol 2021 Oct 12;77(10):1583-1585. Epub 2021 May 12.

Immunology and Pharmacology Laboratory, National Institute for Infectious Diseases "Lazzaro Spallanzani" IRCCS, via Portuense 292, 00149, Rome, Italy.

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http://dx.doi.org/10.1007/s00228-021-03128-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116063PMC
October 2021

PMN-MDSC Frequency Discriminates Active Versus Latent Tuberculosis and Could Play a Role in Counteracting the Immune-Mediated Lung Damage in Active Disease.

Front Immunol 2021 26;12:594376. Epub 2021 Apr 26.

Laboratory of Cellular Immunology and Pharmacology, National Institute for infectious Diseases "Lazzaro Spallanzani"-IRCCS, Rome, Italy.

Tuberculosis (TB), due to infection, is still the principal cause of death caused by a single infectious agent. The balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. Factors defining this variety are unclear and likely involve both mycobacterial and immunological components. Myeloid derived suppressor cells (MDSC) have been shown to be expanded during TB, but their role in human TB pathogenesis is not clear. We evaluated the frequency of circulating MDSC by flow-cytometry in 19 patients with active TB, 18 with latent TB infection (LTBI), and 12 healthy donors (HD) as control. Moreover, we investigated the capacity of MDSC to modulate the mycobactericidal activity of monocytes. The association between MDSC level and TB chest X-ray severity score was analyzed. We observed that, unlike active TB, polymorphonuclear (PMN)-MDSC are not expanded in LTBI patients, and, by performing a receiver operating characteristic (ROC) curve analysis, we found that PMN-MDSC frequency supported the discrimination between active disease and LTBI. Interestingly, we observed an association between PMN-MDSC levels and the severity of TB disease evaluated by chest X-ray. Specifically, PMN-MDSC frequency was higher in those classified with a low/mild severity score compared to those classified with a high severity score. Moreover, PMN-MDSC can impact mycobacterial growth by inducing ROS production in Bacillus Calmette et Guerin (BCG)-infected monocytes. This effect was lost when tested with (MTB), In conclusion, our data indicate that the elevated frequency of PMN-MDSC in IGRA-positive individuals is associated with active TB. Our findings also pointed out a beneficial role of PMN-MDSC during human active TB, most likely associated with the limitation of inflammation-induced tissue damage.
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http://dx.doi.org/10.3389/fimmu.2021.594376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107479PMC
June 2021

No Efficacy of the Combination of Lopinavir/Ritonavir Plus Hydroxychloroquine Versus Standard of Care in Patients Hospitalized With COVID-19: A Non-Randomized Comparison.

Front Pharmacol 2021 22;12:621676. Epub 2021 Apr 22.

National Institute for Infectious Diseases Lazzaro Spallanzani IRCCS, Rome, Italy.

No specific treatment has been approved for COVID-19. Lopinavir/ritonavir (LPV/r) and hydroxychloroquine (HCQ) have been used with poor results, and a trial showed advantages of combined antiviral therapy vs. single antivirals. The aim of the study was to assess the effectiveness of the combination of antivirals (LPV/r and HCQ) or their single use in COVID-19 hospitalized patients vs. standard of care (SoC). Patients ≥18 years with SARS-CoV-2 infection, defined as positive RT-PCR from nasal/oropharyngeal (NP/OP) swab or positive serology, admitted at L. Spallanzani Institute (Italy) were included. time to invasive ventilation/death. Secondary endpoint: time to two consecutive negative SARS-CoV-2 PCRs in NP/OP swabs. In order to control for measured confounders, a marginal Cox regression model with inverse probability weights was used. A total of 590 patients were included in the analysis: 36.3% female, 64 years (IQR 51-76), and 91% with pneumonia. Cumulative probability of invasive ventilation/death at 14 days was 21.2% (95% CI 17.6, 24.7), without difference between SOC, LPV/r, hydroxychloroquine, HCQ + LPV/r, and SoC. The risk of invasive ventilation/death in the groups appeared to vary by baseline ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2). Overall cumulative probability of confirmed negative nasopharyngeal swabs at 14 days was 44.4% (95% CI 38.9, 49.9), without difference between groups. In this retrospective analysis, we found no difference in the rate of invasive ventilation/death or viral shedding by different strategies, as in randomized trials performed to date. Moreover, even the combination HCQ + LPV/r did not show advantages vs. SoC.
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http://dx.doi.org/10.3389/fphar.2021.621676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100580PMC
April 2021

Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

J Immunol 2021 05 3;206(10):2420-2429. Epub 2021 May 3.

National Institute for Infectious Diseases IRCCS "L. Spallanzani," Rome, Italy

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-β production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNβ production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-β production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.
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http://dx.doi.org/10.4049/jimmunol.2001122DOI Listing
May 2021

Prolonged and severe SARS-CoV-2 infection in patients under B-cell-depleting drug successfully treated: A tailored approach.

Int J Infect Dis 2021 Jun 24;107:247-250. Epub 2021 Apr 24.

National Institute for Infectious Diseases 'Lazzaro Spallanzani' IRCCS, via Portuense 292, 00149, Rome, Italy.

Prolonged B-cell depletion due to anti-CD20 monoclonal antibody (mAbs) therapy impairs the adaptive immune response, causing severe manifestations during COronaVIrus Disease-2019 (COVID-19). The cases of two patients under anti-CD20 therapy who experienced prolonged and severe COVID-19 successfully treated with mAbs against Severe Acute Respiratory Syndrome-CoV-2 spike proteins are reported.
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http://dx.doi.org/10.1016/j.ijid.2021.04.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065233PMC
June 2021
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