Impact of Mycobacterium avium subsp. paratuberculosis infection on bovine IL10RA knockout mammary epithelial (MAC-T) cells.

Mycobacterium avium subsp. Paratuberculosis (MAP) is an intracellular pathogen that causes Johne's disease (JD) in cattle and other ruminants. IL10RA encodes the alpha chain of the IL-10 receptor that binds the cytokine IL-10, and is one of the candidate genes that have been found to be associated with JD infection status. In this study, a previously developed IL10RA knockout (IL10RAKO) bovine mammary epithelial (MAC-T) cell line and wild-type (WT) MAC-T cells were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of IL10RA. Cytokine and chemokine concentrations in culture supernatants were measured by multiplexing immunoassay. Total RNA was extracted from the MAC-T cells, and qPCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of TNF-α, IL-6, CXCL8, CXCL10, CCL2, and CCL3 were significantly induced in WT MAC-T cells and IL-10 was significantly inhibited post-MAP infection. However, IL10RAKO MAC-T cells had greater secretion of TNF-α, IL-6, IFN-γ, CCL3, CCL4, CXCL8, and CXCL10, and lower secretion of VEGF-α. Moreover, the expression of inflammatory genes (TNF-α, IL-1α, IL-6) was also more significantly induced in IL10RAKO cells than in WT MAC-T cells post-MAP-infection, and unlike the WT cells, anti-inflammatory cytokines IL-10 and SOCS3 and chemokines CCL2 were not significantly induced. In addition, the expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP-infection; however, there was no significant induction of these miRNAs in the IL10RAKO cells, which suggests IL10 receptor is somehow involved in regulating the miRNA response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in interleukin signaling, and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of IL10RA in the regulation of innate immune response to MAP.

Role of Toll-Like Receptor 4 in Mycobacterium avium subsp. paratuberculosis Infection of Bovine Mammary Epithelial (MAC-T) Cells In Vitro.

Toll-like receptor 4 (TLR4) encodes an innate immune cell pattern-recognition receptor implicated in the recognition of Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in ruminants. Polymorphisms in TLR4 have been associated with susceptibility to MAP infection. In this study, a previously developed TLR4 knockout (TLR4KO) bovine mammary epithelial (MAC-T) cell line and wild-type MAC-T cells (WT) were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of TLR4. Cytokines/chemokines production in culture supernatants was measured by multiplexing immunoassay. Total RNA was extracted from the remaining MAC-T cells, and quantitative PCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), CXCL8, CXCL10, CCL4, and CCL3 were significantly induced in WT MAC-T cells during MAP infection. However, TLR4KO MAC-T cells had greater secretion of CCL3, IL-6, vascular endothelial growth factor (VEGF-α), and TNF-α and decreased secretion of CXCL10 and CCL2. Moreover, the expression of inflammatory genes was induced in TLR4KO cells. The expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP infection; however, there was no significant induction of these miRNAs in TLR4KO cells, which suggests they are involved in regulating the innate immune response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in TLR and interleukin signaling and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of TLR4 in the regulation of innate immune response to MAP. IMPORTANCE Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent for paratuberculosis or Johne's disease (JD) in ruminants, a disease clinically very similar to Crohn's disease in humans. Polymorphisms in the bovine Toll-like receptor genes (TLR1, TLR2, and TLR4) have been shown to affect MAP recognition and host innate immune response and have been associated with increased susceptibility of cattle to paratuberculosis. Our results demonstrated that knocking out the TLR4 gene in bovine MAC-T cells enhanced inflammation in response to MAP. These findings show divergent roles for TLR4 in Escherichia coli lipopolysaccharide and mycobacterial infections, and this may have important consequences for the treatment of these inflammatory diseases and for genetic selection to improve disease resistance. It advances our understanding of the role of TLR4 in the context of MAP infection.

Possible Synergistic Effects of Thymol and Nicotine Against Crithidia bombi Parasitism in Bumble Bees.

Floral nectar contains secondary compounds with antimicrobial properties that can affect not only plant-pollinator interactions, but also interactions between pollinators and their parasites. Although recent work has shown that consumption of plant secondary compounds can reduce pollinator parasite loads, little is known about the effects of dosage or compound combinations. We used the generalist pollinator Bombus impatiens and its obligate gut parasite Crithidia bombi to study the effects of nectar chemistry on host-parasite interactions. In two experiments we tested (1) whether the secondary compounds thymol and nicotine act synergistically to reduce parasitism, and (2) whether dietary thymol concentration affects parasite resistance. In both experiments, uninfected Bombus impatiens were inoculated with Crithidia and then fed particular diet treatments for 7 days, after which infection levels were assessed. In the synergism experiment, thymol and nicotine alone and in combination did not significantly affect parasite load or host mortality. However, the thymol-nicotine combination treatment reduced log-transformed parasite counts by 30% relative to the control group (P = 0.08). For the experiment in which we manipulated thymol concentration, we found no significant effect of any thymol concentration on Crithidia load, but moderate (2 ppm) thymol concentrations incurred a near-significant increase in mortality (P = 0.054). Our results tentatively suggest the value of a mixed diet for host immunity, yet contrast with research on the antimicrobial activity of dietary thymol and nicotine in vertebrate and other invertebrate systems. We suggest that future research evaluate genetic variation in Crithidia virulence, multi-strain competition, and Crithidia interactions with the gut microbe community that may mediate antimicrobial activities of secondary compounds.

Relationships within inpatient physician housestaff teams and their association with hospitalized patient outcomes.

IMPORTANCE: Improving inpatient care delivery has historically focused on improving individual components of the system. Applying the complexity science framework to clinical systems highlights the important role of relationships among providers in influencing system function and clinical outcomes. OBJECTIVE: To understand whether inpatient medical physician teams can be differentiated based on the relationships among team members, and whether these relationships are associated with patient outcomes, including length of stay (LOS), unnecessary length of stay (ULOS), and complication rates. DESIGN: Eleven inpatient medicine teams were observed daily during attending rounds for 2- to 4-week periods from September 2008 through June 2011. Detailed field notes were taken regarding patient care activities, team behaviors, and patient characteristics and outcomes. Behaviors were categorized using the Lanham relationship framework, giving each team a relationship score. We used factor analysis to assess the pattern of relationship characteristics and assessed the association between relationship characteristics and patient outcomes. SETTING: Observations occurred at the Audie L. Murphy Veterans Affairs Hospital and University Hospital in San Antonio, Texas. PARTICIPANTS: Physicians were chosen based on rotation schedules, experience, and time of year. Patients were included based on their admission to the inpatient medicine teams that were being observed. MAIN MEASURES: Relationship scores were based on the presence or absence of 7 relationship characteristics. LOS, ULOS, and complication rates were assessed based on team discussions and chart review. The association between relationships and outcomes was assessed using the Kruskal-Wallis rank sum test. RESULTS: We observed 11 teams over 352.9 hours, observing 1941 discussions of 576 individual patients. Teams exhibited a range of 0 to 7 relationship characteristics. Relationship scores were significantly associated with complication rates, and presence of trust and mindfulness among teams was significantly associated with ULOS and complication rates. CONCLUSIONS: Our findings are an important step in understanding the impact of relationships on the outcomes of hospitalized medical patients. This understanding could expand the scope of interventions to improve hospital care to include not only process improvement but also relationships among providers.