Fabio Filippini

Human anelloviruses are ubiquitous members of the commensal virome, and blood Torquetenovirus (TTV) load has emerged as a candidate marker of host immune status. Their behavior during acute respiratory viral infections across blood and respiratory compartments remains poorly defined. To assess TTV DNA load by quantitative PCR, anellovirus species distribution by next‐generation sequencing, soluble immune markers, and blood‐respiratory genomic overlap of anelloviruses in hospitalized patients with acute respiratory viral infection. Study design: A single‐center observational study was performed on leftover specimens from patients admitted to the University Hospital of Varese, Italy, between September 2024 and March 2025. We collected 37 respiratory specimens and 9 plasma samples. Respiratory viruses were identified by multiplex real‐time PCR. TTV DNA was quantified in blood and respiratory samples by an in‐house universal TaqMan assay targeting the untranslated region; this assay provides a total TTV DNA measurement. Anellovirus species composition was characterized separately by amplicon‐based next‐generation sequencing. Plasma cytokines and adhesion molecules were measured by automated microfluidic immunoassay. In five paired blood‐respiratory cases, reconstructed anellovirus genomes were compared at the whole‐genome level. Total TTV load differed by infecting virus, with significantly higher levels in human rhinovirus (HRV) than influenza (FLU) or respiratory syncytial virus (RSV) in both blood and respiratory samples. IFN‐β was higher in FLU than RSV, whereas ICAM‐1 was higher in HRV than RSV. Age had a stronger effect than sex: children aged 0‐10 years had higher blood and respiratory TTV loads and higher IFN‐α, IFN‐β, and ICAM‐1, whereas adults aged 34–90 years showed greater representation of TTV9 and TTV24. No significant association was found between the number of detected anellovirus species and TTV DNA load. Whole‐genome comparison showed partial overlap of blood‐ and respiratory‐derived anelloviruses without compartment‐specific clustering. In acute respiratory viral infections, TTV DNA load and age‐related differences in anellovirus community composition provided information distinct from simple anellovirus species counts. HRV infection was associated with the highest total TTV burden, and blood and respiratory compartments shared part of the same anellovirus population.

Background/Objectives: Recent studies have shown that GABA reduces visceral hypersensitivity and improves intestinal permeability in a post-inflammatory irritable bowel syndrome (IBS) rat model. We aimed to assess the efficacy of a GABA-based supplement in IBS patients with diarrhea (IBS-D), focusing on symptoms relief, quality-of-life improvement, mucosal barrier function, systemic microinflammation and gut microbiota. Methods: In this double-blind, placebo-controlled, crossover study, 20 IBS-D patients were randomized to receive GABA or placebo for two four-week treatment periods separated by a two-week washout. Efficacy was assessed using IBS Symptom Severity Score (IBS-SSS) and Short-Form Health Survey-36 (SF-36). Circulating levels of lipopolysaccharide-binding protein (LBP), Tumor Necrosis Factor-α (TNF-α) and interleukin (IL)-1β were measured before and after each treatment. Results: Eighteen patients completed the study. A clinical response (≥50-point reduction in IBS-SSS) was observed in 66.7% of patients during GABA treatment versus 33.3% with placebo. GABA produced a significant reduction in the IBS-SSS total score (p = 0.02) and in the bowel satisfaction item of the questionnaire (p = 0.02). Regarding quality of life, GABA significantly improved the “Emotional limitation” domain compared with placebo (p = 0.009). GABA treatment also led to a decrease in circulating LBP (p = 0.06) and IL-1β (p = 0.02) levels compared to placebo, although only the reduction in IL-1β reached statistical significance. In contrast, no substantial remodeling of the gut microbiota was observed. Conclusions: In this pilot study, GABA treatment led to a significant improvement in IBS-D symptoms compared with placebo and was also more effective in enhancing emotional wellbeing. GABA appeared to have a possible effect on intestinal permeability indirectly assessed through LBP, consistent with preclinical findings, and significantly reduced systemic inflammation. GABA may represent a promising therapeutic option for IBS, deserving further investigation in larger clinical trials.

Background: Diabetes-associated neurodegeneration is amplified by methylglyoxal (MGO)-driven dicarbonyl stress linking hyperglycemia to neuronal insulin resistance and maladaptive neuroinflammation. We tested the neuroprotective activity of MNP-021, a non-electrophilic TRPA1 modulator, in neurons and glial cells in vitro . Methods: SH-SY5Y neurons were pretreated with MNP-021 and challenged with MGO, then profiled by high-content imaging, RNA-seq, Seahorse OCR/ECAR, glycolytic stress assays and AKT/ERK/CREB immunoblotting ± insulin. In parallel, HMC3 glial cells were treated with MNP-021, exposed to LPS/TNF-α or Aβ _(25–35) and tested for viability and inflammatory markers by ELISA and qRT-PCR. Results: MGO increased nucleus-to-cytoplasm area ratio by 49% and dysregulated glucose handling, increasing 2-NBDG uptake by ∼25%, with GLUT1/GLUT4 membrane redistribution; MNP-021 normalized morphology, uptake, and transporter localization without cytotoxicity up to 10 µM. RNA-seq identified 754 MGO-deregulated genes, including ISR/metabolic nodes (GCK, SESN2, PHGDH/PSAT1, PCK2); MNP-021 buffered stress-induced transcription with limited baseline effects, remodeled mitochondrial redox readouts consistent with controlled ROS signaling, while improving mitochondrial content/architecture and blunting stress-evoked compensatory glycolysis. MNP-021 restored pro-survival signaling (pAKT/pERK and nuclear pCREB), including insulin responsiveness during MGO exposure. MNP-021 reduced IL-6/TNF-α release while increasing IL-10 and ARG1 (∼1.9-fold vs LPS/TNF-α) in HMC3 glial cells, shifting them toward a pro-resolving IL-10/ARG1 program with reduced Aβ _(25–35)- evoked cytokine release with GLP-1 remaining very low (≤10 pg/mL) and not significantly increased in this system. Conclusions: MNP-021 coordinates transcriptomic restraint, transporter-level glucose handling, mitochondrial resilience, and pro-survival/pro-resolving signaling across neuron–microglia compartments, supporting TRPA1-tuned small-molecule modulation as a candidate strategy against dicarbonyl-linked neuro-metabolic stress.

Human DEAD-box helicase 3 (DDX3) is a multifunctional RNA helicase implicated in mRNA unwinding and the regulation of gene expression. While DDX3 has been extensively studied in the context of RNA virus replication, its role in DNA virus replication remains less understood. In this study, we explore the involvement of DDX3 in the life cycle of Herpes Simplex Virus type 2 (HSV-2), a double-stranded DNA virus. Silencing of DDX3 expression with siRNA significantly impaired HSV-2 replication, indicating that DDX3 supports viral propagation. Unexpectedly, HSV-2 infection led to a marked reduction in cellular DDX3 protein levels during in vitro replication in human cells, particularly at 24 h post-infection, corresponding to the peak of viral production. Notably, this decrease was not accompanied by a reduction in DDX3 mRNA levels, nor was it prevented by proteasome inhibition, suggesting an alternative mechanism of DDX3 depletion. Further analysis revealed substantial amounts of DDX3 protein within HSV-2 virions, supporting the hypothesis that DDX3 is packaged into viral particles during replication. We propose that HSV-2 exploits host DDX3 by incorporating it into progeny virions to facilitate early stages of infection in newly infected cells. However, no evidence linking DDX3 to the assembly process of HSV-2 particles was found. These findings expand the known functional repertoire of DDX3 and highlight its potential as a host factor co-opted by DNA viruses, suggesting a broader relevance in antiviral strategies.

Torque Teno Virus (TTV) is a nonpathogenic and ubiquitous ssDNA virus, a member of the Anelloviridae family. TTV has been postulated as a biomarker in transplant patients. This study aimed to determine the TTV species diversity and variability in renal transplant recipients and to associate species diversity with the corresponding TTV viral load. From 27 recipients, 30 plasma samples were selected. Viral load was determined using two real-time PCR assays, followed by RCA-NGS and ORF1 phylogenetic analysis. The TTV diversity was determined in all samples. Variability was determined in three patients with two sequential samples (pre- and post-transplantation). Most of the samples presented multiple TTV species, up to 15 different species were detected. In the pre-transplant samples (n = 12), the most prevalent species were TTV3 (75%) and TTV13 (75%), and the median number of species per sample was 5 (IQR: 4–7.5). TTV3 was also the most prevalent (56%) in the post-transplant samples (n = 18), and the median number of species was 2 (IQR: 1.8–5.5). No significant correlation between the number of species and viral load was found. The number and type of TTV species showed total variability over time. We report high TTV species diversity in Argentinian recipients, especially in pre-transplant period, with total intra-host variability. However, we found no significant correlation between this high diversity and TTV viral load.

A next-generation sequencing (NGS) study identified a very high viral load of Torquetenovirus (TTV) in KD patients. We aimed to evaluate the feasibility of a newly developed quantitative species-specific TTV-PCR (ssTTV-PCR) method to identify the etiology of KD. We applied ssTTV-PCR to samples collected from 11 KD patients and 22 matched control subjects who participated in our previous prospective study. We used the NGS dataset from the previous study to validate ssTTV-PCR. The TTV loads in whole blood and nasopharyngeal aspirates correlated highly (Spearman’s R = 0.8931, p < 0.0001, n = 33), supporting the validity of ssTTV-PCR. The ssTTV-PCR and NGS results were largely consistent. However, inconsistencies occurred when ssTTV-PCR was more sensitive than NGS, when the PCR primer sequences mismatched the viral sequences in the participants, and when the NGS quality score was low. Interpretation of NGS requires complex procedures. ssTTV-PCR is more sensitive than NGS but may fail to detect a fast-evolving TTV species. It would be prudent to update primer sets using NGS data. With this precaution, ssTTV-PCR can be used reliably in a future large-scale etiological study for KD.