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: Over the past decade, TNF inhibitors such as Infliximab and Adalimumab have become central to Inflammatory Bowel Diseases treatment, greatly enhancing patient outcomes. However, immunogenicity—where anti-drug antibodies diminish effectiveness—remains an issue, often requiring dose changes or combination therapies. Pharmacogenomics is increasingly applied in IBD to personalise treatment, especially since genetic factors like the HLA-DQA1*05 variant heighten the immunogenicity risk with IFX. This study aims to examine the relationship between the HLA-DQA1*05 variant and response loss or antibody development in patients regularly monitored on IFX or ADA. Methods: Sixty-five paediatric IBD patients were enrolled, with therapeutic drug monitoring (TDM) of IFX and ADA, conducted using immunoenzymatic assays. The presence of the HLA-DQA1*05 T>C allele variant was also tested using a Biomole HLA-DQA1 Real-time PCR kit. Results: The HLA-DQA1*05 rs2097432 T>C allele was present in 54% of patients on IFX and 69% of those on ADA. No statistically significant differences were found between HLA carriers and non-carriers across any of the three analysed groups: IFX, ADA and the overall anti-TNFα. Conclusions: Our study suggests that the HLA-DQA1*05 allele does not increase the risk of secondary loss of response to anti-TNF therapy, likely because most patients were on a combination of anti-TNF agents and immunomodulators, which can lower anti-drug antibody production. Testing for HLA-DQA105 can aid in personalising treatment and optimising therapy to minimise immunogenicity risks.

Bronchiolitis is an acute lower respiratory tract infection mainly affecting children under 24 months, primarily caused by Respiratory Syncytial Virus (RSV). Interferons (IFNs), cytokines central to innate and adaptive immunity, are secreted in response to viral infections. In RSV bronchiolitis, Type I and III IFN Signatures modulate antiviral responses and impact disease severity. This study aimed to evaluate the correlation between Type I and III IFNs transcriptional signatures in blood and nasal swabs with clinical presentation. We analyzed transcription levels of Type I and III IFN Signatures using a PCR real-time TaqMan assay in blood and nasal swabs from children hospitalized for RSV bronchiolitis and from healthy controls (HC). Interferon score I was significantly higher in bronchiolitis patients at admission than at discharge and compared to HC, in both blood and nasal swabs (p < 0.0001). A strong positive correlation for IFN score I between the two sample types was found (p < 0.0001). Patients with severe disease (requiring intensive care) had lower IFN score I than those with milder disease, in both blood (p = 0.0005) and nasal swabs (p = 0.0078). IFN score III was down-regulated in bronchiolitis patients compared to HC in blood samples (p < 0.0001), while in nasal swabs, this down-regulation was evident only at discharge (p < 0.0001 vs admission; p = 0.0546 vs HC). This study enhances understanding of IFN signature dynamics in RSV bronchiolitis, emphasizing the importance of sample type and signature specificity, and suggests their potential use as prognostic tools to improve patient management.

Background: Respiratory syncytial virus (RSV) bronchiolitis is a leading cause of lower respiratory tract infections in children under two years of age. NF-κB is a key transcription factor in antiviral and inflammatory responses. This study investigates the expression of NF-κB mRNA in both blood and buccal swab samples of pediatric patients hospitalized for RSV bronchiolitis, comparing levels at admission and discharge. Methods: Paired peripheral blood and buccal swab samples were collected from pediatric patients (n = 85) at hospital admission and discharge. Quantitative real-time PCR was used to assess NF-κB mRNA levels. Results: NF-κB mRNA levels significantly decreased in blood between admission and discharge (p < 0.05), while no significant change was observed in buccal swabs. Conclusions: These results suggest a compartment-specific regulation of NF-κB, with systemic inflammatory resolution at discharge and persistent or distinct mucosal immune activity. Understanding these dynamics may improve our approach to monitoring and treating RSV bronchiolitis.

Systemic lupus erythematosus (SLE) is characterized by chronic immune activation, enhanced type I interferon signaling, and epigenetic dysregulation, conditions that may promote the reactivation of human endogenous retroviruses (HERVs). Whether HERV activation in SLE is global or selective, however, remains unclear. We analyzed the expression of HERV-H, HERV-K, and HERV-W, along with the HERV-derived envelope genes Syncytin-1 and Syncytin-2, in samples from lupus patients and healthy controls. In parallel, we assessed the expression of the epigenetic repressors TRIM28 and SETDB1. HERV-H expression was comparable between groups, whereas HERV-K and HERV-W were significantly overexpressed in lupus patients. Syncytin-1 and HERV-W env transcripts were markedly increased in SLE, while Syncytin-2 expression was unchanged. Lupus patients showed reduced TRIM28 and increased SETDB1 expression, consistent with altered regulation of HERV repression pathways. Notably, HERV-H and HERV-W pol expression correlated with the type I interferon score, suggesting an association between interferon signaling and selective HERV activation. These findings indicate that SLE is associated with the selective activation of immunostimulatory HERV families, particularly HERV-W. The observed associations with interferon signaling suggest that HERV-W-related transcripts may represent disease-associated molecular signatures, warranting further mechanistic investigation.

Background: Fas/FasL-mediated apoptosis is central to immune homeostasis and is implicated in autoimmune lymphoproliferative syndrome (ALPS) and immune thrombocytopenia (ITP). We aimed to compare whole-blood transcriptional levels of CASP8 and CASP3 across ALPS, chronic ITP, and healthy controls. Methods: CASP8 and CASP3 mRNA expression was quantified by real-time PCR in whole blood from clinically diagnosed ALPS patients, chronic ITP patients, and healthy controls. Results: CASP8 mRNA expression was significantly increased in ALPS and ITP versus controls (p = 0.0009 and p < 0.0001, respectively) and was lower in ALPS than in ITP (p = 0.0265). CASP3 mRNA was also increased in both patient groups versus controls (ALPS: p = 0.0045; ITP: p < 0.0001), with no significant difference between ALPS and ITP (p = 0.1692). Conclusions: ALPS and chronic ITP show distinct CASP8 transcriptional patterns and a shared upregulation of CASP3 at the whole-blood mRNA level. These findings are descriptive and do not directly assess caspase activation or apoptotic pathway activity; further protein- and cell subset-based studies are needed to clarify functional implications.