Project description:Background: Farm exposures in early life reduce the risks for childhood allergic diseases and asthma. There is less information about how farm exposures relate to respiratory illnesses and mucosal immune development. Objective: We hypothesized that children raised in farm environments have a lower incidence of viral illnesses over the first two years of life than non-farm children. We also analyzed between farm exposures or respiratory illnesses were related to patterns of nasal cell gene expression. Methods: The Wisconsin Infant Study Cohort (WISC) birth cohort enrolled farm and non-farm pregnant women from central Wisconsin. Parents reported prenatal farm and other environmental exposures. Illness frequency and severity were assessed using illness diaries and periodic surveys. Nasopharyngeal cell gene expression at age two years was compared to farm exposure and respiratory illness history. Results: There was a higher rate of respiratory illnesses in the non-farm vs. farm group (rate ratio 0.82 [0.69,0.97], p=0.020), but no significant differences in wheezing illnesses. There was a stepwise reduction in rates of respiratory illnesses in children exposed at least weekly to 0, 1, or ≥2 animals (p=0.006). In analyzing nasal cell gene expression, farm exposures and preceding respiratory illnesses were positively related to gene signatures for mononuclear cells and innate and antimicrobial responses. Conclusions: Children exposed to farms and farm animals had lower rates of respiratory illnesses over the first two years of life. Both farm exposures and preceding respiratory illnesses were associated with increased innate immune responses, suggesting that these exposures stimulate mucosal immune responses to reduce subsequent illness frequency.
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls.
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls. HEK293 cells (human origin) transiently transfected with 4 various plasmids
Project description:Background: Inhalation exposure to biological particulate matter (BioPM) from livestock farms may provoke exacerbations in subjects suffering from allergy and asthma. The aim of this study was to use a murine model of allergic asthma to determine the effect of BioPM derived from goat farm on airway allergic responses Methods: Fine (< 2.5 μm) BioPM was collected from an indoor goat stable. Female BALB/c mice were ovalbumin (OVA) sensitized and challenged with OVA or saline as control. The OVA and saline groups were divided in sub-groups and exposed intranasally to different concentrations (0, 0.9, 3, or 9 μg) of goat farm BioPM. Bronchoalveolar lavage fluid (BALF), blood and lung tissues were collected. Results: In saline-challenged mice, goat farm BioPM alone induced a dose-dependent increase in neutrophils in BALF and induced production of macrophage inflammatory protein-3a). In OVA-challenged mice, BioPM significantly enhanced 1) inflammatory cells in BALF, 2) OVA-specific Immunoglobulin (Ig)G1, 3) interleukin-23 production, 4) airway mucus secretion-specific gene expression. RNAseq analysis of lungs indicates that neutrophil chemotaxis and oxidation-reduction processes were the representative genomic pathways in saline and OVA-challenged mice, respectively. Conclusions: A single exposure to goat farm BioPM enhanced airway inflammation in both saline and OVA-challenged allergic mice, with neutrophilic response as Th17 disorder and eosinophilic response as Th2 disorder indicative of the severity of allergic responses. Identification of the mode of action by which farm PM interacts with airway allergic pathways will be useful to design potential therapeutic approaches.