Stefano Gambarino

Background: Epstein–Barr virus (EBV) has long been linked to systemic lupus erythematosus (SLE), mainly through molecular mimicry, altered B-cell biology, defective antiviral immune surveillance and intermittent viral reactivation. However, the detection of EBV DNA in clinical samples does not necessarily indicate transcriptionally active infection or direct viral modulation of host pathways. Methods: Peripheral blood samples from 39 pediatric patients with SLE and 29 healthy controls were analyzed for host immune and apoptotic gene expression, including NFKB1, IL10, TLR3, BCL2, BAX and CASP3. EBV DNA was assessed in both patients and controls. In EBV DNA-positive SLE samples, the expression of EBV transcripts representative of latent, lytic and immunomodulatory programs, including LMP1, EBNA1, BZLF1, and viral IL-10, was also evaluated. Host gene expression was additionally compared between EBV DNA-positive and EBV DNA-negative patients with SLE. Results: EBV DNA was detected in 8 of 39 patients with SLE (20.5%) and in none of the 29 healthy controls (0%; Fisher’s exact test, p = 0.0171). None of the EBV DNA-positive samples showed detectable expression of LMP1, EBNA1, BZLF1 or viral IL-10. Compared with healthy controls, patients with SLE showed increased CASP3 expression that remained significant after FDR correction, whereas the reduction in IL10 expression was nominally significant only in the unadjusted analysis. NFKB1, TLR3, BCL2, and BAX did not differ significantly. In the exploratory comparison between EBV DNA-positive and EBV DNA-negative patients with SLE, no host gene remained significantly associated with EBV DNA status after false discovery rate correction. Conclusions: EBV DNA was detected more frequently in patients with SLE than in healthy controls, but this finding was not accompanied by detectable expression of the selected EBV transcripts under the analytical conditions used or by a consistent host transcriptional signature associated with EBV DNA positivity. These findings emphasize the importance of distinguishing EBV DNA detection from detectable viral transcript expression in SLE.

Background: Polymorphisms in the TATA-box of the UGT1A1 promoter are responsible for Gilbert syndrome and play a key role in irinotecan-related toxicity. Reliable, rapid, and cost-effective genotyping methods are therefore required in routine molecular diagnostics. Methods and Results: We performed an analytical validation of a High-Resolution Melting (HRM) assay designed to discriminate UGT1A1 TATA-box alleles. A total of 106 neonatal clinical samples were analyzed to assess genotype distribution under routine diagnostic conditions. Analytical validation was performed using plasmid controls, reference genomic DNA samples, and sequencing-confirmed in-house samples. Plasmid-derived TA5, TA6, TA7, and TA8 genotypes and their heterozygous combinations were tested in quintuplicate. Reference genomic DNA samples carrying rare or non-observed genotypes were tested in triplicate, and 30 in-house samples representative of TA6/TA6, TA6/TA7, and TA7/TA7 genotypes were tested in triplicate and confirmed by Sanger sequencing. The HRM assay discriminated the tested genotypes based on melting temperature (Tm) and curve morphology. HRM genotype calls showed complete concordance with expected or reference genotypes. Intra-test analysis of the TA6/TA7 standard showed a mean Tm of 43.36 °C, SD of 0.20 °C, and CV of 0.46%, while inter-test analysis showed identical Tm values across replicates. Conclusions: The UGT1A1 TATA-box HRM assay showed reproducible genotype discrimination in the tested validation panel and may represent a practical approach for routine UGT1A1 promoter genotyping.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into various connective tissue cell types. Adipose tissue provides a rich source of MSCs (ADSCs), which can differentiate into osteoblasts, adipocytes, and chondroblasts. Pluripotency factors such as SOX2, NANOG, and OCT4 maintain MSC stemness, whereas human endogenous retroviruses (HERVs) and their epigenetic regulators TRIM28 and SETDB1 have been implicated in transcriptional regulation and cell fate decisions. This study investigated the transcriptional expression of HERV-H, -K, and -W, TRIM28, SETDB1, and pluripotency markers (NANOG, OCT4, SOX2) during chondrogenic differentiation of ADSCs using Real-Time PCR. Chondrogenesis was confirmed by aggrecan (ACAN) upregulation and aggrecan immunostaining. Although no statistically significant differences were observed for HERV-H, HERV-K, or HERV-W, HERV-K and HERV-W showed a trend toward decreased expression in differentiated cells, consistent with the overall shift in transcriptional profile during lineage commitment. TRIM28 expression was significantly reduced, while SETDB1 showed a decreasing trend. Among pluripotency markers, OCT4 was significantly downregulated, whereas NANOG and SOX2 remained stable. Correlation analyses revealed that in differentiated ADSCs, HERV-W expression correlated negatively with TRIM28 and positively with SETDB1, while no correlations were found for HERV-H or HERV-K. These findings suggest that specific HERV families and their epigenetic regulators may undergo coordinated modulation during chondrogenic differentiation, supporting a complex and family-specific interplay between retroelement regulation, pluripotency factors, and MSC lineage commitment.

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgene^TM Blood RNA Tubes, Tempus^TM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard^® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at −80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at −80, −20, 4 °C, or room temperature for up to 2 months after, and then could be stored at −80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses.

Background and Objectives: In this study, we applied one-step real time rt-PCR technology type II INF signature to blood and nasopharyngeal (NPS) swabs of acute early recovery children < 1 years hospitalized for bronchiolitis with laboratory-confirmed RSV infection. Materials and Methods: A prospective observational case–control study was conducted in 2021–2022. The study took place in Children Hospital “Regina Margherita”, Torino Italy. The study included 66 infants, of which 30 patients were hospitalized for bronchiolitis due to RSV infection and 36 age-matched controls. Inclusion criteria included a positive RSV test for infants with bronchiolitis. We collected peripheral blood and nasopharyngeal swabs for relative quantification of type II Interferon signature by One-Step Multiplex PCR real time. Results: IFN levels were downregulated in the peripheral blood of bronchiolitis patients; these data were not confirmed in the nasopharyngeal swab. There was no correlation between NPS and the type II IFN score in peripheral blood. Conclusions: our study shows for the first time that type II IFN score was significant reduced in peripheral blood of infants with bronchiolitis by RSV compared to age-matched healthy controls; in the NPS swab this resulted downregulation was not statistically significant and the type II IFN score in the NPS swab can be used as marker of resolution of infection or improvement of clinical conditions.

It has been proven that single-nucleotide polymorphisms (SNPs) in LEP and LEPR genes could predispose individuals to an increased risk of pregnancy adverse outcomes (PAOs) such as recurrent pregnancy loss (RPL) and pre-eclampsia. Preterm birth (PTB) is the leading cause of infant mortality. We decided to investigate the correlation between PTB and LEP and LEPR SNPs. The study cohort included families who underwent spontaneous PTB and control samples of families who had at-term-born (≥37 weeks of gestational age) children. Swabs were performed by rubbing the sticky end for about 30 s on the gum and on the inside of the cheek, allowing us to collect the flaking cells of the oral mucosa. Genotyping of the three SNPs—LEPRA668G, LEPG2548A and A19G—was carried out via an ARMS-MAMA real-time PCR procedure, as previously described. Regarding LEPG2548A, we found that the most expressed genotype in infants both in the preterm and the at-term group was AG; however, we did not discover any statistically significant difference (p = 0.97). Considering LEPA19G, none among the infants and parents were found to carry the AA genotype. No statistically significant differences were found between children, mothers and fathers belonging to preterm and at-term groups. We did not find a statistically significant association in newborns and their mother, but our results show a statistical correlation with the LEPRA668G genotype GG of the father. This fact can contribute to defining genetic risk factors for PTB. Further studies are certainly needed to better clarify the role of genetics in influencing preterm delivery.

Chronic immune thrombocytopenia (CITP) is an autoimmune disease whose underlying biologic mechanisms remain elusive. Human endogenous retroviruses (HERVs) derive from ancestral infections and constitute about 8% of our genome. A wealth of clinical and experimental studies highlights their pivotal pathogenetic role in autoimmune diseases. Epigenetic mechanisms, such as those modulated by TRIM28 and SETDB1, are involved in HERV activation and regulation of immune response. We assessed, through a polymerase chain reaction real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of Syncytin (SYN)1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 in whole blood from 34 children with CITP and age-matched healthy controls (HC). The transcriptional levels of all HERV sequences, with the exception of HERV-W-env, were significantly enhanced in children with CITP as compared to HC. Patients on eltrombopag treatment exhibited lower expression of SYN1, SYN2, and HERV-W-env as compared to untreated patients. The mRNA concentrations of TRIM28 and SETDB1 were significantly higher and were positively correlated with those of HERVs in CITP patients. The over-expressions of HERVs and TRIM28/SETDB1 and their positive correlations in patients with CITP are suggestive clues of their contribution to the pathogenesis of the disease and support innovative interventions to inhibit HERV and TRIM28/SETDB1 expressions in patients unresponsive to standard therapies.

Introduction. MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as antiviral responses, but its role during respiratory syncytial virus (RSV) infections is not known. The objective of this study was to investigate the expression of miR-155 using pharyngeal swabs and peripheral blood in infants with RSV infection and uninfected controls. Methods. A prospective age-matched study was conducted in primary care in Torino from 1 August 2018 to 31 January 2020. We enrolled 66 subjects, 29 of them patients with RSV infection and 37 age-matched uninfected controls, and collected pharyngeal swabs and peripheral blood in order to assess miR-155 expression with real-time stem–loop–TaqMan real-time PCR. Results. The data show that there is no correlation between pharyngeal swabs and peripheral blood with respect to miR-155 expression. The 1/ΔCq miR-155 expression levels in throat swabs in RSV bronchiolitis patients and healthy controls were 0.19 ± 0.11 and 0.21 ± 0.09, respectively, and were not significantly different between healthy controls and bronchiolitis (p = 0.8414). In the peripheral blood, miR-155 levels were higher than those of healthy control subjects: 0.1 ± 0.013 and 0.09 ± 0.0007, respectively; p = 0.0002. Discussion. Our data provide evidence that miR-155 expression is higher in peripheral blood during RSV infection but not in swabs. This difference in the timing of sample recruitment could explain the differences obtained in the results; miR-155 activation is probably only assessable in the very early stages of infection in the swab and remains visible for longer in the blood. New investigations are needed in order to clarify whether the miR-155 expression in swabs can be influenced by different stages of virus disease of infants.

Accumulating evidence highlights the pathogenetic role of human endogenous retroviruses (HERVs) in eliciting and maintaining multiple sclerosis (MS). Epigenetic mechanisms, such as those regulated by TRIM 28 and SETDB1, are implicated in HERV activation and in neuroinflammatory disorders, including MS. Pregnancy markedly improves the course of MS, but no study explored the expressions of HERVs and of TRIM28 and SETDB1 during gestation. Using a polymerase chain reaction real-time Taqman amplification assay, we assessed and compared the transcriptional levels of pol genes of HERV-H, HERV-K, HERV-W; of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis associated retrovirus (MSRV); and of TRIM28 and SETDB1 in peripheral blood and placenta from 20 mothers affected by MS; from 27 healthy mothers, in cord blood from their neonates; and in blood from healthy women of child-bearing age. The HERV mRNA levels were significantly lower in pregnant than in nonpregnant women. Expressions of all HERVs were downregulated in the chorion and in the decidua basalis of MS mothers compared to healthy mothers. The former also showed lower mRNA levels of HERV-K-pol and of SYN1, SYN2, and MSRV in peripheral blood. Significantly lower expressions of TRIM28 and SETDB1 also emerged in pregnant vs. nonpregnant women and in blood, chorion, and decidua of mothers with MS vs. healthy mothers. In contrast, HERV and TRIM28/SETDB1 expressions were comparable between their neonates. These results show that gestation is characterized by impaired expressions of HERVs and TRIM28/SETDB1, particularly in mothers with MS. Given the beneficial effects of pregnancy on MS and the wealth of data suggesting the putative contribution of HERVs and epigenetic processes in the pathogenesis of the disease, our findings may further support innovative therapeutic interventions to block HERV activation and to control aberrant epigenetic pathways in MS-affected patients.