In vitro resistance mechanisms of Neisseria meningitidis against neutrophil extracellular traps

Neisseria meningitidis (Nm) is a leading cause of septicemia in childhood. Nm septicemia is unique with respect to very quick disease progression, high in vivo bacterial replication rate and its considerable mortality. Nm circumvents major mechanisms of innate immunity such as complement system and phagocytosis. Neutrophil extracellular traps (NETs) are formed from neutrophils during systemic infection and are suggested to contain invading microorganisms. Here, we investigated the interaction of Nm with NETs. Both, meningococci and spontaneously released outer membrane vesicles (SOMVs) were potent NET inducers. NETs were unable to kill NET bound meningococci, but slowed down their proliferation rate. Using Nm as model organism we identified three novel mechanisms how bacteria can evade NET‐mediated killing: (i) modification of lipid A of meningococcal LPS with phosphoethanolamine protected Nm from NET‐bound cathepsin G; (ii) expression of the high‐affinity zinc uptake receptor ZnuD allowed Nm to escape NET‐mediated nutritional immunity; (iii) binding of SOMVs to NETs saved Nm from NET binding and the consequent bacteriostatic effect. Escape from NETs may contribute to the most rapid progression of meningococcal disease. The induction of NET formation by Nm in vivo might aggravate thrombosis in vessels ultimately directing to disseminated intravascular coagulation (DIC).     

Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.     

Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

The persistence of Listeria monocytogenes in food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1, cadA2, and cadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated: cadA1 was more common in isolates of serotypes 1/2a and 1/2b than 4b, while cadA2 was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups of L. monocytogenes, including exposures to heavy metals and disinfectants.     

Effect of melatonin on incidence of delirium among patients with hip fracture: a multicentre,double-blind randomized controlled trial

Background:

Disturbance of the sleep–wake cycle is a characteristic of delirium. In addition, changes in melatonin rhythm influence the circadian rhythm and are associated with delirium. We compared the effect of melatonin and placebo on the incidence and duration of delirium.

Methods:

We performed this multicentre, double-blind, randomized controlled trial between November 2008 and May 2012 in 1 academic and 2 nonacademic hospitals. Patients aged 65 years or older who were scheduled for acute hip surgery were eligible for inclusion. Patients received melatonin 3 mg or placebo in the evening for 5 consecutive days, starting within 24 hours after admission. The primary outcome was incidence of delirium within 8 days of admission. We also monitored the duration of delirium.

Results:

A total of 452 patients were randomly assigned to the 2 study groups. We subsequently excluded 74 patients for whom the primary end point could not be measured or who had delirium before the second day of the study. After these postrandomization exclusions, data for 378 patients were included in the main analysis. The overall mean age was 84 years, 238 (63.0%) of the patients lived at home before admission, and 210 (55.6%) had cognitive impairment. We observed no effect of melatonin on the incidence of delirium: 55/186 (29.6%) in the melatonin group v. 49/192 (25.5%) in the placebo group; difference 4.1 (95% confidence interval −0.05 to 13.1) percentage points. There were no between-group differences in mortality or in cognitive or functional outcomes at 3-month follow-up.

Interpretation:

In this older population with hip fracture, treatment with melatonin did not reduce the incidence of delirium. Trial registration: Netherlands Trial Registry, NTR1576: MAPLE (Melatonin Against PLacebo in Elderly patients) study; www.trialregister.nl/trialreg/admin/rctview.asp?TC=1576Delirium in older inpatients is associated with a high risk of dementia and other complications that translate into increased mortality and health care costs.^1,2 The antipsychotic haloperidol has historically been the agent of choice for treating delirium, and it has increasingly been administered as a prophylactic for delirium or to reduce symptoms such as hallucinations and aggressive behaviour.^3,4 However, all antipsychotic treatments may induce serious cerebrovascular adverse effects and greater mortality, particularly among patients with dementia.^5,6 These effects led the US Food and Drug Administration to issue a serious warning against their use.⁷ In addition, benzodiazepines are still frequently used to treat delirium, despite their being known to elicit or aggravate delirium.^8,9Disturbances of the circadian sleep–wake cycle represent one of the core features of delirium,¹⁰ leading to the hypothesis that the neurotransmitter melatonin and changes in its metabolism may be involved in the pathogenesis of delirium.^11,12 Objective measurements have shown that melatonin metabolism is disturbed after abdominal and other types of surgery, insomnia, sleep deprivation and stays in the intensive care unit (ICU), all of which are also known to be factors that contribute to delirium.^13–16 These characteristics suggest an association between melatonin abnormalities and delirium.^17–22 Although proof of a causal relation is still lacking, inpatients might nevertheless benefit from melatonin supplementation therapy through postoperative maintenance or restoration of their sleep–wake cycle.^23–25 Although melatonin depletion is thought to be one of the mechanisms of delirium, few studies have investigated the effects of altering perioperative plasma concentrations of melatonin, in particular, the possible effects on postoperative delirium.The primary objective of this study was to assess the effects of melatonin on the incidence of delirium among elderly patients admitted to hospital as an emergency following hip fracture. Secondary outcomes were duration and severity of delirium, length of hospital stay, total doses of haloperidol and benzodiazepines administered to patients with delirium, mortality during the hospital stay, and functional status, cognitive function and mortality at 3-month follow-up.     

Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding

Microbial motility frequently depends on flagella or type?IV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous with type?IV pili assembly systems of Gram-negative bacteria. Therefore elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the type?II/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. In the present study we show that purified FlaI is a Mn2+-dependent ATPase that binds MANT-ATP [2'-/3'-O-(N'- methylanthraniloyl)adenosine-5'-O-triphosphate] with a high affinity and hydrolyses ATP in a co-operative manner. FlaI has an optimum pH and temperature of 6.5 and 75?°C for ATP hydrolysis. Remarkably, archaeal, but not bacterial, lipids stimulated the ATPase activity of FlaI 3-4-fold. Analytical gel filtration indicated that FlaI undergoes nucleotide-dependent oligomerization. Furthermore, SAXS (small-angle X-ray scattering) analysis revealed an ATP-dependent hexamerization of FlaI in solution. The results of the present study report the first detailed biochemical analyses of the motor protein of an archaeal flagellum.     

Reversible resistance induced by FLT3 inhibition: a novel resistance mechanism in mutant FLT3-expressing cells

Objectives

Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.

Methods

MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.

Results and Conclusions

We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.     

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48]

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [³H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [³H]water. The extremely low contamination of the water soluble phase by [1ß-³H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.