The role of H4 flagella in Escherichia coli ST131 virulence

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug resistant clone associated with urinary tract and bloodstream infections. Most ST131 strains exhibit resistance to multiple antibiotics and cause infections associated with limited treatment options. The largest sub-clonal ST131 lineage is resistant to fluoroquinolones, contains the type 1 fimbriae fimH30 allele and expresses an H4 flagella antigen. Flagella are motility organelles that contribute to UPEC colonisation of the upper urinary tract. In this study, we examined the specific role of H4 flagella in ST131 motility and interaction with host epithelial and immune cells. We show that the majority of H4-positive ST131 strains are motile and are enriched for flagella expression during static pellicle growth. We also tested the role of H4 flagella in ST131 through the construction of specific mutants, over-expression strains and isogenic mutants that expressed alternative H1 and H7 flagellar subtypes. Overall, our results revealed that H4, H1 and H7 flagella possess conserved phenotypes with regards to motility, epithelial cell adhesion, invasion and uptake by macrophages. In contrast, H4 flagella trigger enhanced induction of the anti-inflammatory cytokine IL-10 compared to H1 and H7 flagella, a property that may contribute to ST131 fitness in the urinary tract.

Analysis of human immunodeficiency virus type 1 drug resistance in children receiving nucleoside analogue reverse-transcriptase inhibitors plus nevirapine, nelfinavir, or ritonavir (Pediatric AIDS Clinical Trials Group 377)

In Pediatric AIDS Clinical Trials Group 377, antiretroviral therapy-experienced children were randomized to 4 treatment arms that included different combinations of stavudine, lamivudine (3TC), nevirapine (Nvp), nelfinavir (Nfv), and ritonavir (Rtv). Previous treatment with zidovudine (Zdv), didanosine (ddI), or zalcitabine (ddC) was acceptable. Drug resistance ((R)) mutations were assessed before study treatment (baseline) and at virologic failure. Zdv(R), ddI(R), and ddC(R) mutations were detected frequently at baseline but were not associated with virologic failure. Children with drug resistance mutations at baseline had greater reductions in virus load over time than did children who did not. Nvp(R) and 3TC(R) mutations were detected frequently at virologic failure, and Nvp(R) mutations were more common among children receiving 3-drug versus 4-drug Nvp-containing regimens. Children who were maintained on their study regimen after virologic failure accumulated additional Nvp(R) and 3TC(R) mutations plus Rtv(R) and Nfv(R) mutations. However, Rtv(R) and Nfv(R) mutations were detected at unexpectedly low rates.

GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

Purpose: Gamma-aminobutyric acid A (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i. Methods: Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM. Results: Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM). Conclusions: GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.