Comparative effects of a candidate modified-risk tobacco product Aerosol and cigarette smoke on human organotypic small airway cultures: a systems toxicology approach.

Using an in vitro human small airway epithelium model, we assessed the biological impact of an aerosol from a candidate modified-risk tobacco product, the tobacco heating system (THS) 2.2, to investigate the potential reduced risk of THS2.2 aerosol exposure compared with cigarette smoke. Following the recommendations of the Institute of Medicine and the Tobacco Product Assessment Consortium, in which modified-risk tobacco products assessment should be performed in comparison with standard conventional products, the effects of the THS2.2 aerosol exposure on the small airway cultures were compared with those of 3R4F cigarette smoke. We used a systems toxicology approach whereby elucidation of toxic effects is derived not only from functional assay readouts but also from omics technologies. Cytotoxicity, ciliary beating function, secretion of pro-inflammatory mediators and histological assessment represented functional assays. The omics data included transcriptomic and miRNA profiles. Exposure-induced perturbations of causal biological networks were computed from the transcriptomic data. The results showed that THS2.2 aerosol exposure at the tested doses elicited lower cytotoxicity levels and lower changes in the secreted pro-inflammatory mediators than 3R4F smoke. Although THS2.2 exposure elicited alterations in the gene expression, a higher transcriptome-induced biological impact was observed following 3R4F smoke: The effects of THS2.2 aerosol exposure, if observed, were mostly transient and diminished more rapidly after exposure than those of 3R4F smoke. The study demonstrated that the systems toxicology approach can reveal changes at the cellular level that would be otherwise not detected from functional assays, thus increasing the sensitivity to detect potential toxicity of a treatment/exposure.

Human three-dimensional engineered neural tissue reveals cellular and molecular events following cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common cause of congenital infection of the central nervous system (CNS). To overcome the limited access to human neural tissue and stringent species specificity of HCMV, we used engineered neural tissues to: (i) provide a technical advance to mimick features of HCMV infection in a human neural fetal tissue in vitro and (ii) characterize the molecular and cellular phenomenon following HCMV infection in this tissue. Herein, we infected hESC-derived engineered neural tissues (ENTs) whose organization resembles fetal brain. Transcriptome analysis of ENTs demonstrated that HCMV infection displayed features of the infection with the expression of genes involved in lipid metabolism, growth and development, as well as stress and host-response in a time-dependent manner. Immunohistochemical analysis demonstrated that HCMV did not firstly infect neural tubes (i.e. radially organized, proliferating stem cell niches), but rather an adjacent side population of post-mitotic cells expressing nestin, doublecortin, Sox1, musashi and vimentin markers. Importantly, we observe the same tropism in naturally HCMV-infected fetal brain specimens. To the best of our knowledge this system represents the first human brain-like tissue able to provide a more physiologically model for studying HCMV infection.

Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection.

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.

Cellular diversity within embryonic stem cells: pluripotent clonal sublines show distinct differentiation potential.

Embryonic stem cells (ESC), derived from the early inner cell mass (ICM), are constituted of theoretically homogeneous pluripotent cells. Our study was designed to test this concept using experimental approaches that allowed characterization of progenies derived from single parental mouse ESC. Flow cytometry analysis showed that a fraction of ESC submitted to neural differentiation generates progenies that escape the desired phenotype. Live imaging of individual cells demonstrated significant variations in the capacity of parental ESC to generate neurons, raising the possibility of clonal diversity among ESC. To further substantiate this hypothesis, clonal sublines from ESC were generated by limit dilution. Transcriptome analysis of undifferentiated sublines showed marked differences in gene expression despite the fact that all clones expressed pluripotency markers. Sublines showed distinct differentiation potential, both in phenotypic differentiation assays and with respect to gene expression in embryoid bodies. Clones generated from another ESC line also showed individualities in their differentiation potential, demonstrating the wider applicability of these findings. Taken together, our observations demonstrate that pluripotent ESC consist of individual cell types with distinct differentiation potentials. These findings identify novel elements for the biological understanding of ESC and provide new tools with a major potential for their future in vitro and in vivo use.

Experimental human rhinovirus and enterovirus interspecies recombination.

Human rhinoviruses (HRVs) and enteroviruses (HEVs), two important human pathogens, are non-enveloped, positive-sense RNA viruses of the genus Enterovirus within the family Picornaviridae. Intraspecies recombination is known as a driving force for enterovirus and, to a lesser extent, rhinovirus evolution. Interspecies recombination is much less frequent among circulating strains, and supporting evidence for such recombination is limited to ancestral events, as shown by recent phylogenetic analyses reporting ancient HRV-A/HRV-C, HEV-A/HEV-C and HEV-A/HEV-D recombination mainly at the 5'-untranslated region (5' UTR)-polyprotein junction. In this study, chimeric genomes were artificially generated using the 5' UTR from two different clinical HRV-C strains (HRV-Ca and HRV-Cc), an HRV-B strain (HRV-B37) and an HEV-A strain (HEV-A71), and the remaining part of the genome from an HRV-A strain (HRV-A16). Whilst the chimeric viruses were easily propagated in cell culture, the wild-type HRV-A16 retained a replication advantage, both individually and in competition experiments. Assessment of protein synthesis ability did not show a correlation between translation and replication efficiencies. These results reflect the interchangeability of the 5' UTR, including its functional RNA structural elements implicated in both genome translation and replication among different enterovirus species. The 5' UTR-polyprotein junction therefore represents a theoretic interspecies recombination breakpoint. This recombination potential is probably restricted by the need for co-infection opportunities and the requirement for the progeny chimera to outcompete the parental genomes' fitness, explaining the rare occurrence of such events in vivo.