Virulence factors in urinary Escherichia coli strains: phylogenetic background and quinolone and fluoroquinolone resistance

Quinolone- and fluoroquinolone-resistant Escherichia coli strains harbor fewer virulence factors than susceptible strains. The reasons underlying this correlation are incompletely understood. We investigated the phylogenetic background, the presence of the papC, hlyA, and cnf1 (pathogenicity island II_J96-associated), fimA, iss, and iutA genes, and the presence of type 1 fimbriae, P fimbriae, and hemolysin in 243 urinary E. coli isolates resistant only to quinolones (8%), resistant to both quinolones and fluoroquinolones (51%), or susceptible to both drugs (41%). Group B2 accounted for 56% of the isolates, showing a significantly higher prevalence among fluoroquinolone-susceptible strains than among resistant strains (65% versus 50% [P = 0.03]). hly and cnf1 were significantly more associated with susceptibility (P < 0.001) and with group B2 (P < 0.001 for group B2 versus groups A and D). However, within group B2, fluoroquinolone-resistant strains showed lower prevalences of papC, hlyA, and cnf1 than their susceptible counterparts (P < 0.001). In contrast, the incidence of iutA appeared higher for refractory isolates, including group B2, than for susceptible isolates (P < 0.001). Only in group B2 did fluoroquinolone-resistant strains reveal a lesser ability to agglutinate Saccharomyces cerevisiae (7%) than quinolone-resistant (87%) and susceptible (80%) isolates, despite uniform possession of fimA genes. No similar contrast emerged for expression of hemolysin and P fimbriae. Mutations conferring quinolone and fluoroquinolone resistance may thus require a particular genetic background, not strictly correlated with phylogenetic groups. More interestingly, the mutational event itself can affect the expression of type 1 fimbriae, at least in the prevalent and complex B2 strains.     

Pafah1b2 mutations suppress the development of hydrocephalus in compound Pafah1b1; Reln and Pafah1b1; Dab1 mutant mice

Reelin, an extracellular protein that signals through the Dab1 adapter protein, and Lis1 regulate neuronal migration and cellular layer formation in the brain. Loss of Reelin and reduction in Lis1 activity in mice or humans results in the disorganization of cortical structures. Lis1, the product of the Pafah1b1 gene associates with Alpha1 (the product of the Pafah1b3 gene) and Alpha2 (the product of the Pafah1b2 gene) to form the Pafah1b heterotrimeric complex. This complex interacts biochemically and genetically with the Reelin pathway, however, the role of Alpha1 and Alpha2 in brain development is poorly understood. We previously demonstrated that compound mutations of Pafah1b1 with genes in Reelin pathway result in layering defects and the appearance of hydrocephalus in double mutant mice. Here, we generate triple mouse mutants to investigate the effect of individual Pafah1b Alpha subunits on cellular layer formation and hydrocephalus. We found that Pafah1b3 mutations exacerbate the layering defects, whereas Pafah1b2 mutations strongly suppress the hydrocephalus phenotype of compound mutant mice. The data indicate that the two Pafah1b Alpha subunits have profoundly different effects on brain development and interact in a significantly different manner with the Reelin signaling pathway.     

Analysis of natural killer cells isolated from human decidua: Evidence that 2B4 (CD244) functions as an inhibitory receptor and blocks NK-cell function

While during the first trimester of pregnancy natural killer (NK) cells represent the most abundant lymphocyte population in the decidua, their actual function at this site is still debated. In this study we analyzed NK cells isolated from decidual tissue for their surface phenotype and functional capability. We show that decidual NK (dNK) cells express normal surface levels of certain activating receptors, including NKp46, NKG2D, and 2B4, as well as of killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A inhibitory receptor. In addition, they are characterized by high levels of cytoplasmic granules despite their CD56(bright) CD16- surface phenotype. Moreover, we provide evidence that in dNK cells, activating NK receptors display normal triggering capability whereas 2B4 functions as an inhibitory receptor. Thus, cross-linking of 2B4 resulted in inhibition of both cytolytic activity and interferon-gamma (IFN-gamma) production. Clonal analysis revealed that, in the majority of dNK cell clones, the 2B4 inhibitory function is related to the deficient expression of signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) mRNA. Moreover, biochemical analysis revealed low levels of SAP in the dNK polyclonal population. This might suggest that dNK cells, although potentially capable of killing, are inhibited in their function when interacting with cells expressing CD48.     

Increased serum resistin in nonalcoholic fatty liver disease is related to liver disease severity and not to insulin resistance

CONTEXT: The recently discovered hormone resistin is linked to the development of insulin resistance, but direct evidence of resistin levels in humans with nonalcoholic fatty liver disease (NAFLD) is lacking. METHODS: We conducted this study to assess the relationship between serum resistin and NAFLD. We measured serum resistin and biochemical, hormonal, and histological correlates in 28 NAFLD patients, 33 controls, and 30 obese patients [body mass index (BMI), >30 kg/m2] without NAFLD. RESULTS: Resistin and adiponectin expression were measured in sc adipose tissue by quantitative RT-PCR. Resistin was higher in NAFLD patients compared with controls (5.87 +/- 0.49 vs. 4.30 +/- 0.20 ng/ml; P = 0.002) and obese patients (4.37 +/- 0.27 ng/ml; P = 0.002). Increased resistin mRNA was also found in the adipose tissue of NAFLD patients compared with controls and obese subjects. CONCLUSIONS: Both NAFLD and obese patients had lower adiponectin levels, whereas leptin was increased only in the obese group. No correlation was found between resistin and high-sensitivity C-reactive protein, BMI, homeostasis model assessment, insulin, glucose, transaminases, and lipid values. A positive correlation was found between resistin and histological inflammatory score. These data report increased resistin in NAFLD patients that is related to the histological severity of the disease, but do not support a link between resistin and insulin resistance or BMI in these patients.     

TLR9-mediated signals rescue B-cells from Fas-induced apoptosis via inactivation of caspases

The death receptor, CD95/Fas, serves to eliminate potentially dangerous, self-reactive B cells. Engagement of B-cell receptors (BCR) on mature B-cells mediates the escape from cell death resulting in the activation and expansion of antigen specific clones. In addition to the antigen receptors, the receptors of B-cell activating factor belong to the tumor necrosis factor (TNF) family (BAFFR); moreover, the pattern recognition receptor, TLR9 may also deliver survival signals inhibiting Fas-mediated death of B-cells. Our aim was to compare the mechanism of BCR-induced and the BAFFR- or TLR9-stimulated rescue of B-cells from CD95/Fas-mediated apoptosis. We have found that BAFFR and TLR9 collaborate with BCR to protect B-cells from Fas-induced elimination and the rescue is independent of protein synthesis. The results revealed that the TLR9- and BCR-triggered rescue signals are transmitted through partially overlapping pathways; the protein kinase C (PKC) and the abl kinase induced phosphorylation may inactivate caspases in both CpG and anti-IgG stimulated cells. However, PI3-K activation is crucial upon the BCR driven anti-apoptotic effect, while p38 MAPK-mediated inactivation of caspases seems to play essential role in TLR9-mediated protection against Fas-induced programmed cell death.     

Valosin-containing protein (VCP) mutations in sporadic amyotrophic lateral sclerosis

We recently reported that mutations in the valosin-containing protein (VCP) gene are a cause of 1%-2% of familial amyotrophic lateral sclerosis (ALS) cases, but their role in the pathogenesis of sporadic ALS is unclear. We undertook mutational screening of VCP in 701 sporadic ALS cases. Three pathogenic variants (p.Arg159Cys, p.Asn387Thr, and p.R662C) were found in three U.S. cases, each of whom presented with progressive upper and lower motor neuron signs consistent with definite ALS by El Escorial diagnostic criteria. Our data indicate that VCP mutations may underlie apparently sporadic ALS but account for <1% of this form of disease.     

Involvement of PKCα–MAPK/ERK-phospholipase A2 pathway in the Escherichia coli invasion of brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood–brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA₂s siRNA. cPLA₂ and iPLA₂ enzyme activities and cPLA₂ phosphorylation were stimulated after E. coli incubation and were attenuated by PLA₂, PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.     

Pharmacological Properties of NAI-603, a Well-Tolerated Semisynthetic Derivative of Ramoplanin

NAI-603 is a ramoplanin derivative designed to overcome the tolerability issues of the parent drug as a systemic agent. NAI-603 is highly active against aerobic and anaerobic Gram-positive bacteria, with MICs ranging from 0.008 to 8 μg/ml. MICs were not significantly affected by pH (range, 6 to 8), by inoculum up to 10⁸ CFU/ml, or by addition of 50% human serum. Against staphylococci and enterococci, NAI-603 was rapidly bactericidal, with minimum bactericidal concentration (MBC)/MIC ratios never exceeding 4. The frequency of spontaneous resistance was low at 2× to 4× MIC (≤1 × 10⁻⁶ to ≤1 × 10⁻⁸) and below the detection limit (about ≤1 × 10⁻⁹) at 8× MIC. Serial subcultures at 0.5× MIC yielded at most an 8-fold increase in MICs. In a systemic infection induced by methicillin-resistant Staphylococcus aureus (MRSA), the 50% effective dose (ED₅₀) of intravenous (i.v.) NAI-603 was 0.4 mg/kg, lower than that of oral (p.o.) linezolid (1.4 mg/kg) and subcutaneous (s.c.) teicoplanin (1.4 mg/kg) or vancomycin (0.6 mg/kg). In neutropenic mice infected with vancomycin-resistant enterococci (VRE), the ED₅₀s for NAI-603 were 1.1 to 1.6 mg/kg i.v., compared to 0.5 mg/kg i.v. of ramoplanin. The bactericidal activity was confirmed in vivo in the rat granuloma pouch model induced by MRSA, where NAI-603, at 40 mg/kg i.v., induced about a 2- to 3-log₁₀-reduction in viable bacteria in the exudates, which persisted for more than 72 h. The pharmacokinetic (PK) profiles of NAI-603 and ramoplanin at 20 mg/kg show similar half-lives (3.27 and 3.80 h, respectively) with the maximum concentration (C_max) markedly higher for NAI-603 (207 μg/ml versus 79 μg/ml). The favorable pharmacological profile of NAI-603, coupled with the absence of local tolerability issues, supports further investigation.