Bacterial small RNAs may mediate immune response differences seen in respiratory syncytial virus versus rhinovirus bronchiolitis.

Bronchiolitis, a viral lower respiratory infection, is the leading cause of infant hospitalization, which is associated with an increased risk for developing asthma later in life. Bronchiolitis can be caused by several respiratory viruses, such as respiratory syncytial virus (RSV), rhinovirus (RV), and others. It can also be caused by a solo infection (e.g., RSV- or RV-only bronchiolitis) or co-infection with two or more viruses. Studies have shown viral etiology-related differences between RSV- and RV-only bronchiolitis in the immune response, human microRNA (miRNA) profiles, and dominance of certain airway microbiome constituents. Here, we identified bacterial small RNAs (sRNAs), the prokaryotic equivalent to eukaryotic miRNAs, that differ between infants of the 35th Multicenter Airway Research Collaboration (MARC-35) cohort with RSV- versus RV-only bronchiolitis. We first derived reference sRNA datasets from cultures of four bacteria known to be associated with bronchiolitis (i.e., Haemophilus influenzae, Moraxella catarrhalis, Moraxella nonliquefaciens, and Streptococcus pneumoniae). Using these reference sRNA datasets, we found several sRNAs associated with RSV- and RV-only bronchiolitis in our human nasal RNA-Seq MARC-35 data. We also determined potential human transcript targets of the bacterial sRNAs and compared expression of the sRNAs between RSV- and RV-only cases. sRNAs are known to downregulate their mRNA target, we found that, compared to those associated with RV-only bronchiolitis, sRNAs associated with RSV-only bronchiolitis may relatively activate the IL-6 and IL-8 pathways and relatively inhibit the IL-17A pathway. These data support that bacteria may be contributing to inflammation differences seen in RSV- and RV-only bronchiolitis, and for the first time indicate that the potential mechanism in doing so may be through bacterial sRNAs.

Epigenome-wide association analysis of infant bronchiolitis severity: a multicenter prospective cohort study.

Bronchiolitis is the most common lower respiratory infection in infants, yet its pathobiology remains unclear. Here we present blood DNA methylation data from 625 infants hospitalized with bronchiolitis in a 17-center prospective study, and associate them with disease severity. We investigate differentially methylated CpGs (DMCs) for disease severity. We characterize the DMCs based on their association with cell and tissues types, biological pathways, and gene expression. Lastly, we also examine the relationships of severity-related DMCs with respiratory and immune traits in independent cohorts. We identify 33 DMCs associated with severity. These DMCs are differentially methylated in blood immune cells. These DMCs are also significantly enriched in multiple tissues (e.g., lung) and cells (e.g., small airway epithelial cells), and biological pathways (e.g., interleukin-1-mediated signaling). Additionally, these DMCs are associated with respiratory and immune traits (e.g., asthma, lung function, IgE levels). Our study suggests the role of DNA methylation in bronchiolitis severity.

Bacteriophage Therapy for Pan-Drug-Resistant Pseudomonas aeruginosa in Two Persons With Cystic Fibrosis.

Cystic fibrosis (CF) is an important monogenic disease that affects more than 70 000 people worldwide. Defects of the CF transmembrane conductance regulator gene lead to dehydrated viscous secretions that result in chronic bacterial colonization. This leads to frequent recurrent lung infections called pulmonary exacerbations, lung inflammation, and resulting structural lung damage called bronchiectasis. Pseudomonas aeruginosa in particular is a common pathogen in persons with CF associated with increased pulmonary exacerbations, long-term lung function decline, and reduced survival. In addition, P. aeruginosa commonly develops antibiotic resistance and forms biofilms, making it difficult to treat. Here, we report the details of two patients with CF with pan-drug-resistant P. aeruginosa who were treated with a novel therapeutic strategy, bacteriophages. These cases highlight the need for further research and development of this treatment modality, including pediatric clinical trials.

Nasal airway microRNA profiling of infants with severe bronchiolitis and risk of childhood asthma: a multicentre prospective study.

BACKGROUND: Severe bronchiolitis (i.e. bronchiolitis requiring hospitalisation) during infancy is a major risk factor for childhood asthma. However, the exact mechanism linking these common conditions remains unclear. We examined the longitudinal relationship between nasal airway miRNAs during severe bronchiolitis and the risk of developing asthma. METHODS: In a 17-centre prospective cohort study of infants with severe bronchiolitis, we sequenced their nasal microRNA at hospitalisation. First, we identified differentially expressed microRNAs (DEmiRNAs) associated with the risk of developing asthma by age 6 years. Second, we characterised the DEmiRNAs based on their association with asthma-related clinical features, and expression level by tissue and cell types. Third, we conducted pathway and network analyses by integrating DEmiRNAs and their mRNA targets. Finally, we investigated the association of DEmiRNAs and nasal cytokines. RESULTS: In 575 infants (median age 3 months), we identified 23 DEmiRNAs associated with asthma development (e.g. hsa-miR-29a-3p; false discovery rate (FDR) <0.10), particularly in infants with respiratory syncytial virus infection (FDR for the interaction <0.05). These DEmiRNAs were associated with 16 asthma-related clinical features (FDR <0.05), e.g. infant eczema and corticosteroid use during hospitalisation. In addition, these DEmiRNAs were highly expressed in lung tissue and immune cells (e.g. T-helper cells, neutrophils). Third, DEmiRNAs were negatively correlated with their mRNA targets (e.g. hsa-miR-324-3p/IL13), which were enriched in asthma-related pathways (FDR <0.05), e.g. toll-like receptor, PI3K-Akt and FcɛR signalling pathways, and validated by cytokine data. CONCLUSION: In a multicentre cohort of infants with severe bronchiolitis, we identified nasal miRNAs during illness that were associated with major asthma-related clinical features, immune response, and risk of asthma development.

Porcine oocytes matured in a chemically defined medium are transcriptionally active.

The statement that fully-grown porcine oocytes (oocytes from follicles with diameter from 3 to 6 mm) are transcriptionally quiescent is not as strongly supported as it was before. Currently, we know that there is a difference between the transcription profile of germinal vesicle (GV) and metaphase II (MII) oocytes. The goal of our study was to compare the transcription profile of GV, germinal vesicle breakdown (GVBD), metaphase I (MI), and MII oocytes matured in the chemically defined medium FLI. Oocytes were sequenced, and the results were subsequently validated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). We detected multiple differentially transcribed mRNAs, of which many were upregulated. Among them we found mRNAs necessary for protein production, mitochondrial functions and cytoplasmic maturation. Collectively, these data support the hypothesis that transcription activity in fully-grown porcine oocytes is necessary for key processes during their successful maturation in vitro in a chemically defined maturation medium.

Therapeutic beta-lactam dosages and broad-spectrum antibiotics are associated with reductions in microbial richness and diversity in persons with cystic fibrosis.

Persons with cystic fibrosis (PwCF) suffer from pulmonary exacerbations (PEx) related in part to lung infection. While higher microbial diversity is associated with higher lung function, the data on the impact of short-term antibiotics on changes in microbial diversity is conflicting. Further, Prevotella secretes beta-lactamases, which may influence recovery of lung function. We hypothesize that sub-therapeutic and broad spectrum antibiotic exposure leads to decreasing microbial diversity. Our secondary aim was to evaluate the concerted association of beta-lactam pharmacokinetics (PK), antibiotic spectrum, microbial diversity, and antibiotic resistance on lung function recovery using a pathway analysis. This was a retrospective observational study of persons with CF treated with IV antibiotics for PEx between 2016 and 2020 at Children's National Hospital; respiratory samples and clinical information were collected at hospital admission for PEx (E), end of antibiotic treatment (T), and follow-up (F). Metagenomic sequencing was performed; PathoScope 2.0 and AmrPlusPlus were used for taxonomic assignment of sequences to bacteria and antibiotic resistance genes (ARGs). M/W Pharm was used for PK modeling. Comparison of categorical and continuous variables and pathway analysis were performed in STATA. Twenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years. Only 12/43 beta-lactam courses had therapeutic PK, and 18/43 were broad spectrum. A larger decrease in richness between E and T was seen in the therapeutic PK group (sufficient - 20.1 vs. insufficient - 1.59, p = 0.025) and those receiving broad spectrum antibiotics (broad - 14.5 vs. narrow - 2.8, p = 0.030). We did not detect differences in the increase in percent predicted forced expiratory volume in one second (ppFEV1) at end of treatment compared to PEx based on beta-lactam PK (sufficient 13.6% vs. insufficient 15.1%) or antibiotic spectrum (broad 11.5% vs. narrow 16.6%). While both therapeutic beta-lactam PK and broad-spectrum antibiotics decreased richness between PEx and the end of treatment, we did not detect longstanding changes in alpha diversity or an association with superior recovery of lung function compared with subtherapeutic PK and narrow spectrum antimicrobials.

Bioinformatic and experimental methods to identify and validate bacterial RNA-human RNA interactions.

Ample evidence supports the importance of the microbiota on human health and disease. Recent studies suggest that extracellular vesicles are an important means of bacterial-host communication, in part via the transport of small RNAs (sRNAs). Bacterial sRNAs have been shown to co-precipitate with human and mouse RNA-induced silencing complex, hinting that some may regulate gene expression as eukaryotic microRNAs do. Bioinformatic tools, including those that can incorporate an sRNA's secondary structure, can be used to predict interactions between bacterial sRNAs and human messenger RNAs (mRNAs). Validation of these potential interactions using reproducible experimental methods is essential to move the field forward. This review will cover the evidence of interspecies communication via sRNAs, bioinformatic tools currently available to identify potential bacterial sRNA-host (specifically, human) mRNA interactions, and experimental methods to identify and validate those interactions.

Impact of Antibiotics on the Lung Microbiome and Lung Function in Children With Cystic Fibrosis 1 Year After Hospitalization for an Initial Pulmonary Exacerbation.

Background: Cystic fibrosis (CF) is characterized by recurrent pulmonary exacerbations (PEx) and lung function decline. PEx are frequently treated with antibiotics. However, little is known about the effects of antibiotics on the airway microbiome of persons with CF over time. The purpose of this study was to evaluate changes in the microbiome and lung function in persons with CF over 1 year following an initial study pulmonary exacerbation (iPEx). Methods: Twenty children aged ≤18 years with CF were enrolled in the study, which occurred prior to the routine administration of highly effective modulator therapy. Respiratory samples and spirometry were obtained at a minimum of quarterly visits and up to 1 year after an iPEx. Metagenomic sequencing was performed, and bacterial taxa were assigned using MetaPhlAn 2.0. Paired t test, analysis of variance, and generalized least squares regression were used to compare outcome variables. Results: The mean age of study participants at the time of the iPEx was 10.6 years. There were 3 ± 1.6 PEx treated with antibiotics per person during the study period. Bacterial richness was similar at 1 year compared to iPEx (40.3 vs 39.3, P = .852), whereas the mean Shannon diversity index was significantly higher at 1 year (2.84 vs 1.62, P < .001). The number of PEx treated with antibiotics was not associated with changes in microbial diversity but was associated with changes in lung function. Conclusions: In our 1-year prospective study, we found that microbial diversity increased despite decreases in lung function associated with repeated PEx events requiring antibiotic therapy.

Trial of Prasinezumab in Early-Stage Parkinson's Disease.

BACKGROUND: Aggregated α-synuclein plays an important role in the pathogenesis of Parkinson's disease. The monoclonal antibody prasinezumab, directed at aggregated α-synuclein, is being studied for its effect on Parkinson's disease. METHODS: In this phase 2 trial, we randomly assigned participants with early-stage Parkinson's disease in a 1:1:1 ratio to receive intravenous placebo or prasinezumab at a dose of 1500 mg or 4500 mg every 4 weeks for 52 weeks. The primary end point was the change from baseline to week 52 in the sum of scores on parts I, II, and III of the Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS; range, 0 to 236, with higher scores indicating greater impairment). Secondary end points included the dopamine transporter levels in the putamen of the hemisphere ipsilateral to the clinically more affected side of the body, as measured by 123I-ioflupane single-photon-emission computed tomography (SPECT). RESULTS: A total of 316 participants were enrolled; 105 were assigned to receive placebo, 105 to receive 1500 mg of prasinezumab, and 106 to receive 4500 mg of prasinezumab. The baseline mean MDS-UPDRS scores were 32.0 in the placebo group, 31.5 in the 1500-mg group, and 30.8 in the 4500-mg group, and mean (±SE) changes from baseline to 52 weeks were 9.4±1.2 in the placebo group, 7.4±1.2 in the 1500-mg group (difference vs. placebo, -2.0; 80% confidence interval [CI], -4.2 to 0.2; P = 0.24), and 8.8±1.2 in the 4500-mg group (difference vs. placebo, -0.6; 80% CI, -2.8 to 1.6; P = 0.72). There was no substantial difference between the active-treatment groups and the placebo group in dopamine transporter levels on SPECT. The results for most clinical secondary end points were similar in the active-treatment groups and the placebo group. Serious adverse events occurred in 6.7% of the participants in the 1500-mg group and in 7.5% of those in the 4500-mg group; infusion reactions occurred in 19.0% and 34.0%, respectively. CONCLUSIONS: Prasinezumab therapy had no meaningful effect on global or imaging measures of Parkinson's disease progression as compared with placebo and was associated with infusion reactions. (Funded by F. Hoffmann-La Roche and Prothena Biosciences; PASADENA ClinicalTrials.gov number, NCT03100149.).

Nasopharyngeal airway dual-transcriptome of infants with severe bronchiolitis and risk of childhood asthma: A multicenter prospective study.

BACKGROUND: Severe bronchiolitis (ie, bronchiolitis requiring hospitalization) during infancy is a major risk factor for childhood asthma. However, the exact mechanism linking these common conditions remains unclear. OBJECTIVES: This study sought to examine the integrated role of airway microbiome (both taxonomy and function) and host response in asthma development in this high-risk population. METHODS: This multicenter prospective cohort study of 244 infants with severe bronchiolitis (median age, 3 months) examined the infants' nasopharyngeal metatranscriptomes (microbiomes) and transcriptomes (hosts), as well as metabolomes at hospitalization. The longitudinal relationships investigated include (1) major bacterial species (Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis), (2) microbial function, and (3) host response with risks of developing asthma by age 6 years. RESULTS: First, the abundance of S pneumoniae was associated with greater risks of asthma (P = .01), particularly in infants with nonrhinovirus infection (Pinteraction = .04). Second, of 328 microbial functional pathways that are differentially enriched by asthma development, the top pathways (eg, fatty acid and glycolysis pathways; false discovery rate [FDR] < 1 × 10-12) were driven by these 3 major species (eg, positive association of S pneumoniae with glycolysis; FDR < 0.001). These microbial functional pathways were validated with the parallel metabolome data. Third, 104 transcriptome pathways were differentially enriched (FDR < .05)-for example, downregulated interferon-α and -γ and upregulated T-cell activation pathways. S pneumoniae was associated with most differentially expressed transcripts (eg, DAGLB; FDR < 0.05). CONCLUSIONS: By applying metatranscriptomic, transcriptomic, and metabolomic approaches to a multicenter cohort of infants with bronchiolitis, this study found an interplay between major bacterial species, their function, and host response in the airway, and their longitudinal relationship with asthma development.

Measuring the impact of an empiric antibiotic algorithm for pulmonary exacerbation in children and young adults with cystic fibrosis.

BACKGROUND: Antimicrobial stewardship is a systematic effort to change prescribing attitudes that can provide benefit in the provision of care to persons with cystic fibrosis (CF). Our objective was to decrease the unwarranted use of broad-spectrum antibiotics and assess the impact of an empiric antibiotic algorithm using quality improvement methodology. METHODS: We assembled a multidisciplinary team with expertise in CF. We assessed baseline antibiotic use for treatment of pulmonary exacerbation (PEx) and developed an algorithm to guide empiric antibiotic therapy. We included persons with CF admitted to Children's National Hospital for treatment of PEx between January 2017 and March 2020. Our primary outcome measure was reducing unnecessary broad-spectrum antibiotic use, measured by use consistent with the empiric antibiotic algorithm. The primary intervention was the initiation of the algorithm. Secondary outcomes included documentation of justification for broad-spectrum antibiotic use and use of infectious disease (ID) consult. RESULTS: Data were collected from 56 persons with CF who had a total of 226 PEx events. The mean age at first PEx was 12 (SD 6.7) years; 55% were female, 80% were white, and 29% were Hispanic. After initiation of the algorithm, the proportion of PEx with antibiotic use consistent with the algorithm increased from 46.2% to 79.5%. Documentation of justification for broad-spectrum antibiotics increased from 56% to 85%. Use of ID consults increased from 17% to 54%. CONCLUSION: Antimicrobial stewardship initiatives are beneficial in standardizing care and fostering positive working relationships between CF pulmonologists, ID physicians, and pharmacists.

Using metabolic potential within the airway microbiome as predictors of clinical state in persons with cystic fibrosis.

Introduction: Pulmonary exacerbations (PEx) in persons with cystic fibrosis (CF) are primarily related to acute or chronic inflammation associated with bacterial lung infections, which may be caused by several bacteria that activate similar bacterial genes and produce similar by-products. The goal of our study was to perform a stratified functional analysis of bacterial genes at three distinct time points in the treatment of a PEx in order to determine the role that specific airway microbiome community members may play within each clinical state (i.e., PEx, end of antibiotic treatment, and follow-up). Our secondary goal was to compare the change between clinical states with the metabolic activity of specific airway microbiome community members. Methods: This was a prospective observational study of persons with CF treated with intravenous antibiotics for PEx between 2016 and 2020 at Children's National Hospital. Demographic and clinical information as well as respiratory samples were collected at hospital admission for PEx, end of antibiotic treatment, and follow-up. Metagenomic sequencing was performed; MetaPhlAn3 and HUMANn3 were used to assign sequences to bacterial species and bacterial metabolic genes, respectively. Results: Twenty-two persons with CF, with a mean age of 14.5 (range 7-23) years, experienced 45 PEx during the study period. Two-hundred twenty-one bacterial species were identified in the respiratory samples from the study cohort. Ten bacterial species had differential gene abundance across changes in the clinical state including Staphylococcus aureus, Streptococcus salivarius, and Veillonella atypica (all padj < 0.01 and log2FoldChange > |2|). These corresponded to a differential abundance of bacterial genes, with S. aureus accounting for 81% of the genes more abundant in PEx and S. salivarius accounting for 83% of the genes more abundant in follow-up, all compared to the end of treatment. Lastly, 8,653 metabolic pathways were identified across samples, with again S. aureus and S. salivarius contributing to the differential abundance of pathways (106 in PEx vs. 66 in follow-up, respectively). V. atypica was associated with a single metabolic pathway (UDP-N-acetyl-D-glucosamine biosynthesis) increased in follow-up compared to PEx. Discussion: Taken together, these data suggest that the metabolic potential of bacterial species can provide more insight into changes across clinical states than the relative abundance of the bacteria alone.

Integrative genetics-metabolomics analysis of infant bronchiolitis-childhood asthma link: A multicenter prospective study.

Background: Infants with bronchiolitis are at high risk for developing childhood asthma. While genome-wide association studies suggest common genetic susceptibilities between these conditions, the mechanisms underlying the link remain unclear. Objective: Through integrated genetics-metabolomics analysis in this high-risk population, we sought to identify genetically driven metabolites associated with asthma development and genetic loci associated with both these metabolites and asthma susceptibility. Methods: In a multicenter prospective cohort study of infants hospitalized for bronchiolitis, we profiled the nasopharyngeal metabolome and genotyped the whole genome at hospitalization. We identified asthma-related metabolites from 283 measured compounds and conducted metabolite quantitative trait loci (mtQTL) analyses. We further examined the mtQTL associations by testing shared genetic loci for metabolites and asthma using colocalization analysis and the concordance between the loci and known asthma-susceptibility genes. Results: In 744 infants hospitalized with bronchiolitis, 28 metabolites (e.g., docosapentaenoate [DPA], 1,2-dioleoyl-sn-glycero-3-phosphoglycerol, sphingomyelin) were associated with asthma risk. A total of 349 loci were associated with these metabolites-161 for non-Hispanic white, 120 for non-Hispanic black, and 68 for Hispanics. Of these, there was evidence for 30 shared loci between 16 metabolites and asthma risk (colocalization posterior probability ≥0.5). The significant SNPs within loci were aligned with known asthma-susceptibility genes (e.g., ADORA1, MUC16). Conclusion: The integrated genetics-metabolomics analysis identified genetically driven metabolites during infancy that are associated with asthma development and genetic loci associated with both these metabolites and asthma susceptibility. Identifying these metabolites and genetic loci should advance research into the functional mechanisms of the infant bronchiolitis-childhood asthma link.

Nasopharyngeal metatranscriptome profiles of infants with bronchiolitis and risk of childhood asthma: a multicentre prospective study.

BACKGROUND: Bronchiolitis is not only the leading cause of hospitalisation in US infants but also a major risk factor for asthma development. Growing evidence supports clinical heterogeneity within bronchiolitis. Our objectives were to identify metatranscriptome profiles of infant bronchiolitis, and to examine their relationship with the host transcriptome and subsequent asthma development. METHODS: As part of a multicentre prospective cohort study of infants (age <1 year) hospitalised for bronchiolitis, we integrated virus and nasopharyngeal metatranscriptome (species-level taxonomy and function) data measured at hospitalisation. We applied network-based clustering approaches to identify metatranscriptome profiles. We then examined their association with the host transcriptome at hospitalisation and risk for developing asthma. RESULTS: We identified five metatranscriptome profiles of bronchiolitis (n=244): profile A: virusRSVmicrobiomecommensals; profile B: virusRSV/RV-Amicrobiome H.influenzae ; profile C: virusRSVmicrobiome S.pneumoniae ; profile D: virusRSVmicrobiome M.nonliquefaciens ; and profile E: virusRSV/RV-Cmicrobiome M.catarrhalis . Compared with profile A, profile B infants were characterised by a high proportion of eczema, Haemophilus influenzae abundance and enriched virulence related to antibiotic resistance. These profile B infants also had upregulated T-helper 17 and downregulated type I interferon pathways (false discovery rate (FDR) <0.005), and significantly higher risk for developing asthma (17.9% versus 38.9%; adjusted OR 2.81, 95% CI 1.11-7.26). Likewise, profile C infants were characterised by a high proportion of parental asthma, Streptococcus pneumoniae dominance, and enriched glycerolipid and glycerophospholipid metabolism of the microbiome. These profile C infants had an upregulated RAGE signalling pathway (FDR <0.005) and higher risk of asthma (17.9% versus 35.6%; adjusted OR 2.49, 95% CI 1.10-5.87). CONCLUSIONS: Metatranscriptome and clustering analysis identified biologically distinct metatranscriptome profiles that have differential risks of asthma.

MUC1 ectodomain is a flagellin-targeting decoy receptor and biomarker operative during Pseudomonas aeruginosa lung infection.

We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Melatonin improves rate of monospermic fertilization and early embryo development in a bovine IVF system.

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10-11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode's Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10-11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

Generation and first characterization of TRDC-knockout pigs lacking γδ T cells.

The TRDC-locus encodes the T cell receptor delta constant region, one component of the γδ T cell receptor which is essential for development of γδ T cells. In contrast to peptide recognition by αß T cells, antigens activating γδ T cells are mostly MHC independent and not well characterized. Therefore, the function of γδ T cells and their contribution to protection against infections is still unclear. Higher numbers of circulating γδ T cells compared to mice, render the pig a suitable animal model to study γδ T cells. Knocking-out the porcine TRDC-locus by intracytoplasmic microinjection and somatic cell nuclear transfer resulted in healthy living γδ T cell deficient offspring. Flow cytometric analysis revealed that TRDC-KO pigs lack γδ T cells in peripheral blood mononuclear cells (PBMC) and spleen cells. The composition of the remaining leucocyte subpopulations was not affected by the depletion of γδ T cells. Genome-wide transcriptome analyses in PBMC revealed a pattern of changes reflecting the impairment of known or expected γδ T cell dependent pathways. Histopathology did not reveal developmental abnormalities of secondary lymphoid tissues. However, in a vaccination experiment the KO pigs stayed healthy but had a significantly lower neutralizing antibody titer as the syngenic controls.

Impact of Anaerobic Antibacterial Spectrum on Cystic Fibrosis Airway Microbiome Diversity and Pulmonary Function.

BACKGROUND: The role of anaerobic organisms in the cystic fibrosis (CF) lung microbiome is unclear. Our objectives were to investigate the effect of broad (BS) versus narrow (NS) spectrum antianaerobic antibiotic activity on lung microbiome diversity and pulmonary function, hypothesizing that BS antibiotics would cause greater change in microbiome diversity without a significant improvement in lung function. METHODS: Pulmonary function tests and respiratory samples were collected prospectively in persons with CF before and after treatment for pulmonary exacerbations. Treatment antibiotics were classified as BS or NS. Gene sequencing data from 16S rRNA were used for diversity analysis and bacterial genera classification. We compared the effects of BS versus NS on diversity indices, lung function and anaerobic/aerobic ratios. Statistical significance was determined by multilevel mixed-effects generalized linear models and mixed-effects regression models. RESULTS: Twenty patients, 6-20 years of age, experienced 30 exacerbations. BS therapy had a greater effect on beta diversity than NS therapy when comparing time points before antibiotics to after and at recovery. After antibiotics, the NS therapy group had a greater return toward baseline forced expiratory volume at 1 second and forced expiratory flow 25%-75% values than the BS group. The ratio of anaerobic/aerobic organisms showed a predominance of anaerobes in the NS group with aerobes dominating in the BS group. CONCLUSIONS: BS antianaerobic therapy had a greater and possibly longer lasting effect on the lung microbiome of persons with CF, without achieving the recovery of pulmonary function seen with the NS therapy. Specific antibiotic therapies may affect disease progression by changing the airway microbiome.