Informed interpretation of metagenomic data by StrainPhlAn enables strain retention analyses of the upper airway microbiome.

IMPORTANCE: The usage of 16S rRNA gene sequencing has become the state-of-the-art method for the characterization of the microbiota in health and respiratory disease. The method is reliable for low biomass samples due to prior amplification of the 16S rRNA gene but has limitations as species and certainly strain identification is not possible. However, the usage of metagenomic tools for the analyses of microbiome data from low biomass samples is not straight forward, and careful optimization is needed. In this work, we show that by validating StrainPhlAn 3 results with the data from bacterial cultures, the strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA being present when parameters are carefully optimized to fit low biomass microbiomes. This work further proposes that strain retention analyses are feasible, at least for more abundant species. This will help to better understand the longitudinal dynamics of the upper respiratory microbiome during health and disease.

The baseline immunological and hygienic status of pigs impact disease severity of African swine fever.

African Swine Fever virus (ASFV) is a large double-enveloped DNA virus of the Asfarviridae family that causes a lethal hemorrhagic disease in domestic pigs and wild boars. Since 2007, a highly virulent genotype II strain has emerged and spread in Europe and South-East Asia, where millions of animals succumbed to the disease. Field- and laboratory-attenuated strains of ASFV cause highly variable clinical disease severity and survival, and mechanisms remain unclear. We hypothesized that the immunological and hygienic status of pigs is a determinant of ASF disease course. Here we compared the immunological profile at baseline and in response to ASFV infection in specific pathogen-free (SPF) and farm-raised Large White domestic pigs. At steady state, SPF pigs showed lower white blood cell counts and a lower basal inflammatory and antiviral transcriptomic profile compared to farm pigs, associated with profound differences in gut microbiome composition. After inoculation with a highly virulent ASFV genotype II strain (Armenia 2008), severe clinical signs, viremia and pro-inflammatory cytokines appeared sooner in SPF pigs, indicating a reduced capacity to control early virus replication. In contrast, during infection with an attenuated field isolate (Estonia 2014), SPF pigs presented a milder and shorter clinical disease with full recovery, whereas farm pigs presented severe protracted disease with 50% lethality. Interestingly, farm pigs showed higher production of inflammatory cytokines, whereas SPF pigs produced more anti-inflammatory IL-1ra early after infection and presented a stronger expansion of leukocytes in the recovery phase. Altogether, our data indicate that the hygiene-dependent innate immune status has a double-edge sword impact on immune responses in ASF pathogenesis. While the higher baseline innate immune activity helps the host in reducing initial virus replication, it promotes immunopathological cytokine responses, and delays lymphocyte proliferation after infection with an attenuated strain. Such effects should be considered for live vaccine development and vigilance.

Influence of pig farming on human Gut Microbiota: role of airborne microbial communities.

It has been hypothesized that both genetics and diet influence the composition of the human cecal microbiota. However, it remains unclear whether and how occupational exposure to microbes impacts the microbial communities in human guts. Using a One Health approach, we visited pig farms (n = 26) and collected stool specimens from pig workers (n = 59), pig barn air samples (n = 19), and rectal swabs from pigs at three different growth stages (n = 144). Stool samples from cattle workers were included as a control group (n = 22). Each sample's microbiota was characterized using 16S rRNA gene sequencing and the DADA2 pipeline.We obtained a significantly different clustering of the microbial compositions of pig and cattle workers by permutational multivariate analysis of variance (PERMANOVA; P < .001). Workers primarily exposed to pigs had higher relative abundances of Prevotellaceae and less Bacteroidaceae than workers exposed to cattle. We also found that the microbial compositions of pig workers' stool samples shared extensive fractions with the samples from their pigs. We also identified amplicon sequencing variants (ASVs) in the airborne microbiota which were likely involved in zoonotic transmission events.We hypothesize that ASVs originating from pig feces are aerosolized and, through breathing, get trapped in the pig farm workers' upper respiratory tract from where they can get swallowed. Consequently, some of the animal associated ASVs are transferred into the gastrointestinal tracts (GITs) which leads to changes in the composition of the human gut microbiota. The importance of this finding for human health must be investigated further.

Mixed Metal Oxide Nanoparticle Formulations for the Treatment of Seroma.

Seroma formation is a well-recognized postoperative complication for many plastic and general surgical procedures. Although various tissue adhesives and substances have been used in an effort to treat seroma formation, no therapies have been established clinically. Recently, the nano-bridging phenomenon has been introduced as a promising approach to achieve tissue adhesion and strong closure of deep skin wounds in rats. The present study seeks to assess the potential of nano-bridging beyond skin wounds in a rat model of seroma. Seromas were induced in 20 Lewis rats through bilateral axillary lymphadenectomy, excision of the latissimus dorsi and cutaneous maximus muscles, and disruption of dermal lymphatics. On postoperative day (POD) 7, the seroma was aspirated on both sides. A bioactive nanoparticle (NP) suspension based on zinc-doped strontium-substituted bioglass/ceria nanoparticles (NP group) or fibrin glue (fibrin group) was injected into the right seroma cavity, while the left side was left untreated. On POD 14, the NP group showed complete remission (no seromas at all), while the fibrin group recorded a reduction of only 63% in the seroma fluid volume. The NPs exerted local anti-inflammatory and neo-angiogenic effects, without any detectable systemic changes. Moreover, the ceria levels recorded in the organs did not surpass the background level, indicating that the nanoparticles stayed at the site of application. This study is a promising first example demonstrating the ability of inorganic nanoparticle formulations to reduce seroma formation in a rat model, without any detectable systemic adverse effects. These results emphasize the potential of nanotechnological solutions in the therapeutic management of seroma in the clinical setting.

Chronic cigarette smoke exposure and pneumococcal infection induce oropharyngeal microbiota dysbiosis and contribute to long-lasting lung damage in mice.

Environmental factors, such as cigarette smoking or lung infections, may influence chronic obstructive pulmonary disease (COPD) progression by modifying the respiratory tract microbiome. However, whether the disease itself induces or maintains dysbiosis remains undefined. In this longitudinal study, we investigated the oropharyngeal microbiota composition and disease progression of mice (in cages of 5-10 mice per cage) before, during and up to 3 months after chronic cigarette smoke exposure or exposure to room air for 6 months. Cigarette smoke exposure induced pulmonary emphysema measurable at the end of exposure for 6 months, as well as 3 months following smoke exposure cessation. Using both classical culture methods and 16S rRNA sequencing, we observed that cigarette smoke exposure altered the relative composition of the oropharyngeal microbiota and reduced its diversity (P <0.001). More than 60 taxa were substantially reduced after 6 months of smoke exposure (P <0.001) However, oropharyngeal microbiota disordering was reversed 3 months after smoke exposure cessation and no significant difference was observed compared to age-matched control mice. The effects of lung infection with Streptococcus pneumoniae on established smoke-induced emphysema and on the oropharyngeal microbiota were also evaluated. Inoculation with S. pneumoniae induced lung damage and altered the microbiota composition for a longer time compared to control groups infected but not previously exposed to smoke (P=0.01). Our data demonstrate effects of cigarette smoke and pneumococcus infection leading to altered microbiota and emphysema development. The reversal of the disordering of the microbiota composition, but not lung damage, following smoke exposure cessation and after clearance of infection suggest that changes in lung structure are not sufficient to sustain a disordered microbiota in mice. Whether changes in the airway microbiota contribute to inducing emphysema requires further investigation.

Development of vascularized nerve scaffold using perfusion-decellularization and recellularization.

INTRODUCTION: Vascularized nerve grafts (VNG) may offer an advantage in peripheral nerve regeneration by avoiding ischemic damage and central necrosis observed in non-VNG, particularly for the treatment of large and long nerve defects. However, surgical complexity, donor site morbidity and limited nerve availability remain important drawbacks for the clinical use of VNG. Here we explore the potential of perfusion-decellularization for bioengineering a VNG to be used in peripheral nerve reconstruction. METHODS: Porcine sciatic nerves were surgically procured along with their vascular pedicle attached. The specimens were decellularized via perfusion-decellularization and preservation of the extracellular matrix (ECM), vascular patency and tissue cytokine contents were examined. Scaffold reendothelialization was conducted with porcine aortic endothelial cells in a perfusion-bioreactor. RESULTS: Morphologic examination of decellularized VNG and analysis of the DNA content demonstrated cell clearance whereas ECM content and structures of the nerve fascicles were preserved. Using 3D micro-computed tomography imaging we observed optimal vasculature preservation in decellularized scaffolds, down to the capillary level. Cytokine quantification demonstrated measurable levels of growth factors after decellularization. Endothelial cell engraftment of the large caliber vessels was observed in reendothelialized scaffolds. CONCLUSIONS: In this study we provide evidence that perfusion-decellularization can be used to create vascularized nerve scaffolds in which the vasculature and the ECM component are well preserved. As compared to non-vascularized conduits, engineered vascularized nerve scaffolds may represent an ideal approach for promoting better nerve regeneration in larger nerve defect reconstructions.

Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.

INTRODUCTION: Human ear reconstruction is recognized as the emblematic enterprise in tissue engineering. Up to now, it has failed to reach human applications requiring appropriate tissue complexity along with an accessible vascular tree. We hereby propose a new method to process human auricles in order to provide a poorly immunogenic, complex and vascularized ear graft scaffold. METHODS: 12 human ears with their vascular pedicles were procured. Perfusion-decellularization was applied using a SDS/polar solvent protocol. Cell and antigen removal was examined by histology and DNA was quantified. Preservation of the extracellular matrix (ECM) was assessed by conventional and 3D-histology, proteins and cytokines quantifications. Biocompatibility was assessed by implantation in rats for up to 60 days. Adipose-derived stem cells seeding was conducted on scaffold samples and with human aortic endothelial cells whole graft seeding in a perfusion-bioreactor. RESULTS: Histology confirmed cell and antigen clearance. DNA reduction was 97.3%. ECM structure and composition were preserved. Implanted scaffolds were tolerated in vivo, with acceptable inflammation, remodeling, and anti-donor antibody formation. Seeding experiments demonstrated cell engraftment and viability. CONCLUSIONS: Vascularized and complex auricular scaffolds can be obtained from human source to provide a platform for further functional auricular tissue engineered constructs, hence providing an ideal road to the vascularized composite tissue engineering approach. STATEMENT OF SIGNIFICANCE: The ear is emblematic in the biofabrication of tissues and organs. Current regenerative medicine strategies, with matrix from donor tissues or 3D-printed, didn't reach any application for reconstruction, because critically missing a vascular tree for perfusion and transplantation. We previously described the production of vascularized and cell-compatible scaffolds, from porcine ear grafts. In this study, we ---- applied findings directly to human auricles harvested from postmortem donors, providing a perfusable matrix that retains the ear's original complexity and hosts new viable cells after seeding. This approach unlocks the ability to achieve an auricular tissue engineering approach, associated with possible clinical translation.