Network analysis reveals age- and virus-specific circuits in nasal epithelial cells of extremely premature infants.

BACKGROUND AND OBJECTIVES: Viral respiratory infections significantly affect young children, particularly extremely premature infants, resulting in high hospitalization rates and increased health-care burdens. Nasal epithelial cells, the primary defense against respiratory infections, are vital for understanding nasal immune responses and serve as a promising target for uncovering underlying molecular and cellular mechanisms. METHODS: Using a trans-well pseudostratified nasal epithelial cell system, we examined age-dependent developmental differences and antiviral responses to influenza A and respiratory syncytial virus through systems biology approaches. RESULTS: Our studies revealed differences in innate-receptor repertoires, distinct developmental pathways, and differentially connected antiviral network circuits between neonatal and adult nasal epithelial cells. Consensus network analysis identified unique and shared cellular-viral networks, emphasizing highly relevant virus-specific pathways, independent of viral replication kinetics. CONCLUSION: This research highlights the importance of nasal epithelial cells in innate antiviral immune responses and offers crucial insights that allow for a deeper understanding of age-related differences in nasal epithelial cell immunity following respiratory virus infections.

An optimized, robust and reproducible protocol to generate well-differentiated primary nasal epithelial models from extremely premature infants.

Extremely premature infants are prone to severe respiratory infections, and the mechanisms underlying this exceptional susceptibility are largely unknown. Nasal epithelial cells (NEC) represent the first-line of defense and adult-derived ALI cell culture models show promising results in mimicking in vivo physiology. Therefore, the aim of this study was to develop a robust and reliable protocol for generating well-differentiated cell culture models from NECs of extremely premature infants. Nasal brushing was performed in 13 extremely premature infants at term corrected age and in 11 healthy adult controls to obtain NECs for differentiation at air-liquid interface (ALI). Differentiation was verified using imaging and functional analysis. Successful isolation and differentiation was achieved for 5 (38.5%) preterm and 5 (45.5%) adult samples. Preterm and adult ALI-cultures both showed well-differentiated morphology and ciliary function, however, preterm cultures required significantly longer cultivation times for acquiring full differentiation (44 ± 3.92 vs. 23 ± 1.83 days; p < 0.0001). Moreover, we observed that recent respiratory support may impair successful NECs isolation. Herewithin, we describe a safe, reliable and reproducible method to generate well-differentiated ALI-models from NECs of extremely premature infants. These models provide a valuable foundation for further studies regarding immunological and inflammatory responses and respiratory disorders in extremely premature infants.

The PE-PGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins?

A clone was isolated by screening of a cosmid library of Mycobacterium tuberculosis with an oligonucleotide designed from the N-terminal sequence of a previously reported proline-rich protein. Characterization of the 4481 bp insert showed the presence of polymorphic CG-repetitive sequences (PGRSs) with an ORF of 2.7 kb, encoding a 81.3 kDa protein (PE-PGRS81). Southern blot analysis and BLAST-p searches revealed several homologous sequences in the genome of M. tuberculosis. The deduced amino acid sequence was highly similar to a stretch of about 98 residues in the N-terminus present in several members of the PE-PGRS family available in the GenBank database, including 100% identity with the partial amino acid sequence of the potential protein encoded by orf3' as well as with the Rv0278c sequence. A neighbour-joining analysis of the 99 PE-PGRS sequences available in the database indicated that PE-PGRS81 is included in a group where its closest relatives are the sequences orf3', Rv0278c, Rv0279c, Rv1759c, Rv3652 and Rv0747. Probing with the complete coding regions of PE-PGRS81 and Rv1759c in Southern blot assays, on samples of genomic DNA from M. tuberculosis H37Rv, Mycobacterium bovis BCG and M. tuberculosis clinical isolates, showed a complex hybridization pattern for all strains. This shows the existence of intrastrain PGRS variability as reported for other PGRS members. In contrast, probing with the short conserved N-terminal region of Rv1759c reduced the hybridization to a single band. This marker allowed identification of M. tuberculosis clinical strains that lack Rv1759c. A recombinant C-terminal fragment of Rv1759c showed fibronectin-binding properties and was recognized by sera from patients infected with M. tuberculosis, suggesting that at least this member of the PE-PGRS is expressed in tuberculosis infection.