Carla Selvaggi

Differential expression of interferon-induced microRNAs in patients with chronic hepatitis C virus infection treated with pegylated interferon alpha

There have been reports of in-vitro interferon (IFN)-mediated antiviral activity against the hepatitis C virus (HCV) through microRNAs (miRNAs). The main aim of this study was to evaluate the expression of several miRNAs (miR-1, miR-30, miR-128, miR-196, miR-296) in peripheral blood mononuclear cells (PBMCs) from healthy individuals after in vitro IFN-treatment and in PBMCs from patients with chronic hepatitis C (CHC) before and 12 hours after the first injection of pegylated IFN alpha. We demonstrated that expression of these miRNAs could be recorded in PBMCs collected from healthy individuals before and after in-vitro IFN alpha treatment. Our analysis revealed that the levels of expression of all miRNAs investigated in patients with CHC were different to those in healthy individuals. When levels of the miRNAs were measured 12 hours after the first IFN injection, increases in expression levels of IFN-induced miRNAs were observed in 25-50% of patients, depending on the type of miRNA examined. No correlations were observed between HCV viral load, alanine aminotransferase status and expression of miRNA. Together these findings suggest that: (i) IFN alpha in-vitro treatment of PBMCs leads to a transcriptional induction of all miRNAs investigated; (ii) miRNAs can be induced differentially by IFN treatment in patients with HCV. Given the importance of miRNAs in defending the host against virus infections, it is possible that IFN-induced miRNAs may represent an important determinant of the clinical outcome of IFN therapy in HCV infection.

Evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus

The relationship between viral load, disease severity and antiviral immune activation in infants suffering from respiratory syncytial virus (RSV)-associated bronchiolitis has not been well identified. The main objective of this study was to determine the existence of a correlation between RSV load and disease severity and also between different clinical markers and mRNA levels of the interferon stimulated gene (ISG)56 in infants hospitalized for bronchiolitis. We also evaluated whether viral load tended to be persistent over the course of the RSV infection. The levels of RSV-RNA were quantified in nasopharyngeal washings, collected from 132 infants infected with RSV as a single (90.15%) or as a dual infection with other respiratory viruses (9.85%). Results indicated that viral load was positively related to the clinical severity of bronchiolitis, the length of hospital stay, the levels of glycemia and the relative gene expression of ISG56, whereas an inverse correlation was observed with the levels of hemoglobin. We also found that the RSV load significantly decreased between the first and second nasopharingeal washings sample in most subjects. These results suggest that infants with high RSV load on hospital admission are more likely to have both more severe bronchiolitis and a higher airway activation of antiviral immune response.

Gene Expression of Nucleic Acid-Sensing Pattern Recognition Receptors in Children Hospitalized for Respiratory Syncytial Virus-Associated Acute Bronchiolitis

ABSTRACT Given the critical role of pattern recognition receptors (PRRs) in acid nucleic recognition in the initiation of innate immunity and the orchestration of adaptive immunity, the aim of this study was to determine whether any heterogeneity of PRR expression in the airway tracts of infants with respiratory syncytial virus (RSV) infection might explain the broad clinical spectrum of RSV-associated bronchiolitis in infants. For this purpose, the levels of melanoma differentiation-associated protein-5 (MDA-5), retinoic acid inducible gene-1 (RIG-1), and Toll-like receptor 3 (TLR-3), TLR-7, TLR-8, and TLR-9 mRNAs were evaluated, using TaqMan quantitative reverse transcription-PCR, in cells from nasopharyngeal washes collected from 157 infants suffering from acute bronchiolitis whether or not they were associated with respiratory viruses. High interindividual variability was observed in both virus-positive and -negative infants; however, the relative gene expression levels of MDA-5, RIG-1, TLR-7, and TLR-8 were significantly higher in the virus-infected group, whereas the expression levels of TLR-3 and TLR-9 were not significantly different. The differences in the gene expression of MDA-5, RIG-1, TLR-7, and TLR-8 were more evident in infants with RSV infection than in those with bocavirus or rhinovirus infection. In RSV-infected infants, PRR-mRNA levels also were analyzed in relation to interferon protein levels, viral load, clinical severity, days of hospitalization, age, and body weight. A significant positive correlation was observed only between RSV viral load and RIG-1 mRNA levels. These findings provide the first direct evidence that, in infants with respiratory virus-associated bronchiolitis, especially RSV, there are substantial changes in PRR gene expression; this likely is an important determinant of the clinical outcome of bronchiolitis.

Interferon lambda 1–3 expression in infants hospitalized for RSV or HRV associated bronchiolitis

Summary Objectives The airway expression of type III interferons (IFNs) was evaluated in infants hospitalized for respiratory syncytial virus (RSV) or rhinovirus (HRV) bronchiolitis. As an additional objective we sought to determine whether a different expression of IFN lambda 1–3 was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established IFN stimulated genes (ISGs), such as mixovirus resistance A (MxA) and ISG56. Methods The analysis was undertaken in 118 infants with RSV or HRV bronchiolitis. Nasopharyngeal washes were collected for virological studies and molecular analysis of type III IFN responses. Results RSV elicited higher levels of IFN lambda subtypes when compared with HRV. A similar expression of type III IFN was found in RSVA or RSVB infected infants and in those infected with HRVA or HRVC viruses. Results also indicate that IFN lambda 1 and IFN lambda 2–3 levels were correlated with each other and with MxA and ISG56-mRNAs. In addition, a positive correlation exists between the IFN lambda1 levels and the clinical score index during RSV infection. In particular, higher IFN lambda 1 levels are associated to an increase of respiratory rate. Conclusions These findings show that differences in the IFN lambda 1–3 levels in infants with RSV or HRV infections are present and that the expression of IFN lambda 1 correlates with the severity of RSV bronchiolitis.

Expression levels of TLRs involved in viral recognition in PBMCs from HIV-1-infected patients failing antiretroviral therapy.

Objective: The aim of this study was to evaluate whether gene expression of Toll-like receptor (TLR)-3, TLR4, TLR7 and TLR9 was impaired in patients with chronic HIV-1 infection who were failing to respond to antiretroviral therapy. Methods: Transcripts encoding TLRs were assayed by quantitative real time RT-PCR on peripheral blood mononuclear cells derived from patients with HIV-1 infection who were responding or not responding to antiretroviral therapy and healthy control subjects. Results: Chronic HIV-1-infected patients who failed to respond to therapy showed reduced expression of TLRs 3, 4 and 9, together with increased expression of TLR7, as compared to healthy subjects. Moreover, a trend towards a higher expression of TLR3 and TLR9 was observed in responder patients compared with non-responders. In addition, we found lower levels of TRLs 3, 7 and 9 in patients with high levels of HIV-1 RNA compared to those with lower levels of viremia. Conclusions: These findings demonstrate that in chronic HIV-1-positive patients who were failing to respond to the therapy, there were substantial changes in TLRs expression. This is likely to be an important determinant of the clinical course of HIV-1 infection.

Probiotic supplementation promotes a reduction in T-cell activation, an increase in Th17 frequencies, and a recovery of intestinal epithelium integrity and mitochondrial morphology in ART-treated HIV-1-positive patients

HIV infection is characterized by a persistent immune activation associated to a compromised gut barrier immunity and alterations in the profile of the fecal flora linked with the progression of inflammatory symptoms. The effects of high concentration multistrain probiotic (Vivomixx®, Viale del Policlinico 155, Rome, Italy in EU; Visbiome®, Dupont, Madison, Wisconsin in USA) on several aspects of intestinal immunity in ART‐experienced HIV‐1 patients was evaluated.

Measurement of the sensitivity of different commercial assays in the diagnosis of CMV infection in pregnancy.

To evaluate the performance of different commercial assays for the detection of recent cytomegalovirus (CMV) in pregnancy, the sensitivity and specificity of assays for CMV-specific IgM antibodies were compared. Routine specimens from pregnant women were screened for CMV IgM using the Abbott AxSYM assay. Sera that were reactive according to AxSYM were further tested for IgM by other commercial assays. In selected IgM positive samples a CMV IgG avidity assay (Radim) and virus isolation from urine (shell vial) were also performed. The positivity rate for IgM anti-CMV by AxSYM was relatively high (140 out of 492, combining reactive and grayzone results). Only 26 of the 140 samples were positive for IgM according to Radim. The IgG avidity was low in 16 of the 43 samples tested, and the Radim and DiaSorin IgM assays were negative in 5 of them; 2 of the latter cases were also positive for viral isolation according to a shell vial method. There are differences in the sensitivity of the commercially available tests for CMV antibodies. CMV screening in pregnancy is performed as a first step by immunoassays and the choice of highly sensitive IgM test associated with further serological and virological methods could help to identify early primary infections.

Interferon lambda 1 expression in cervical cells differs between low‑risk and high‑risk human papillomavirus‑positive women

Persistent infection by high-risk (HR) human papillomavirus (HPV) types is a prerequisite for progression to cancer. HR-HPVs may lead to a deregulation of innate immunity by interfering with the epithelial type I interferon (IFN) response, whereas very little is known about type III IFNs, a key component of the mucosal antiviral response. This study reports a first attempt to evaluate the activation of type III IFN genes (IFN lambda 1–3), IFN lambda receptor genes (IFN-lambdaR1 and IL10R2), and IFN-induced genes (MxA, ISG15, ISG56) in HPV-positive and HPV-negative cervical cells from 154 women attending the gynecological unit of a university hospital in Rome. Despite an increased individual variability, a coordinated expression of several IFN lambda-related genes was observed. Furthermore, IFN lambda 1 and IFN-lambdaR1 genes were expressed at higher levels in cervical cells positive to low-risk (LR) HPV compared to HR-HPV and HPV-negative cells. Consistently, ISG15 expression was significantly higher in LR-HPV-infected women than in the other groups. Moreover, IFN lambda 1 expression decreased significantly with abnormal cytological results. This study is the first to show the activation of a type III IFN response in LR-HPV-positive cervical cells and suggests that the lack of this response in HR-HPV infection may be related to lesion progression.

ISG15 expression correlates with HIV-1 viral load and with factors regulating T cell response

Given the multifactorial nature of action of type I interferon (IFN) in HIV-1 infection and the need to firmly establish the action of key components of IFN pathways, we compared the IFN stimulated gene (ISG)15 expression with that of other well-characterized ISGs, evaluating its relationship with immunosuppressive factors regulating T-cell response in HIV-1 patients. PBMC from 225 subjects were included: healthy donors (n=30), naïve (n=93) and HAART treated HIV-1 subjects (n=102). Levels of ISG15-mRNA, ISG56-mRNA, APOBEC3G/3F-mRNA, TRAIL-mRNA, IDO-mRNA, proviral load andISG15 (rs15842 and rs1921) SNPs were evaluated by using TaqMan assays. We found that ISG15, ISG56, APOBEC3G/3F levels were increased in untreated HIV-1 patients compared to healthy donors, being ISG15 the highest ISG expressed. The amount of ISG15 correlated with viral load and with CD4+ T cell counts whereas no relationship was found between all ISGs analyzed and proviral load or HIV-1 tropism. ISG15 expression was reduced following long-term antiretroviral therapy. In addition, ISG15 levels were correlated with those of TRAIL and IDO in HIV-1 viremic patients. Lastly, ISG15 SNPs had no influence on ISG15 levels. We demonstrates that ISG15 is elevated in viremic HIV-1 patients and is associated with high TRAIL and IDO levels.

In Vitro Sensitivity of Human Metapneumovirus to Type I Interferons

Human metapneumovirus (hMPV) has been recognized as an important respiratory pathogen. Due to its relatively recent discovery, only limited information is available on the relationship between hMPV and type I interferons (IFN). This study was designed to determine whether in vitro hMPV is sensitive to the antiviral activity of IFN-β, leukocyte IFN-α, and several IFN-α subtypes in a human Hep-2 cell line. The results showed that 50% inhibitory concentration values against hMPV for the various type I IFN preparations were significantly higher than those against the IFN-sensitive vesicular stomatitis virus, and some IFN-α subtypes appeared to be more active against hMPV than others, with IFN-α subtypes 5, 6, 8, and 10 being the most potent, and IFN-α2, 17, and 21 the least potent. The results show that hMPV grown in Hep-2 is partially resistant to the antiviral activity of type I IFNs. Additional studies are required to understand whether and to what extent the relatively low sensitivity of hMPV to IFNs influences the clinical outcomes of infected individuals.