European society of intensive care medicine study of therapeutic hypothermia (32-35°C) for intracranial pressure reduction after traumatic brain injury (the Eurotherm3235Trial)

Background

Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.

Methods/Design

This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.

Discussion

Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.

Trial Registration

ClinicalTrials.gov Identifier: NCT01255215     

Elderly subjects have a delayed antibody response and prolonged viraemia following yellow fever vaccination: a prospective controlled cohort study

Background

Yellow fever vaccination (YF-17D) can cause serious adverse events (SAEs). The mechanism of these SAEs is poorly understood. Older age has been identified as a risk factor. We tested the hypothesis that the humoral immune response to yellow fever vaccine develops more slowly in elderly than in younger subjects.

Method

We vaccinated young volunteers (18–28 yrs, N = 30) and elderly travelers (60–81 yrs, N = 28) with YF-17D and measured their neutralizing antibody titers and plasma YF-17D RNA copy numbers before vaccination and 3, 5, 10, 14 and 28 days after vaccination.

Results

Ten days after vaccination seroprotection was attained by 77% (23/30) of the young participants and by 50% (14/28) of the elderly participants (p = 0.03). Accordingly, the Geometric Mean Titer of younger participants was higher than the GMT of the elderly participants. At day 10 the difference was +2.9 IU/ml (95% CI 1.8–4.7, p = 0.00004) and at day 14 +1.8 IU/ml (95% CI 1.1–2.9, p = 0.02, using a mixed linear model. Viraemia was more common in the elderly (86%, 24/28) than in the younger participants (60%, 14/30) (p = 0.03) with higher YF-17D RNA copy numbers in the elderly participants.

Conclusions

We found that elderly subjects had a delayed antibody response and higher viraemia levels after yellow fever primovaccination. We postulate that with older age, a weaker immune response to yellow fever vaccine allows the attenuated virus to cause higher viraemia levels which may increase the risk of developing SAEs. This may be one piece in the puzzle of the pathophysiology of YEL-AVD.

Trial Registration

Trialregitser.nl NTR1040     

Syntheses and characterization of neutral complexes of Zn(II) with mono- and dianionic pyrrole-2-imine ligands [(pyrrole-2-CHN)nR]n− (n = 1, 2)

The reactions of the dianionic [(pyrrole-2-CHN)₂R]^2? ligands [(N′₂N₂)^2?] (R = (R)(S)-1,2-cyclohexane or 1,2-ethane) with Zn(II) yield neutral dimeric [Zn₂(N′₂N₂)₂] complexes. The dimeric nature of the complexes was established by field-desorption mass spectrometry. ¹H NMR studies show that these complexes have dimeric structures in solution in which the (N′₂N₂)^2? ligands act as di-bidentates.The metal centres have tetrahedral geometries and bot have Δ or Λ configurations. The complex with the (R)(S)-1,2-cyclohexanediyl bridges has a rigid structure in solution. Neither intermolecular nor intramolecular exchange processes are observed The ¹H NMR spectrum of the complex with the 1,2-ethanediyl bridging groups shows that at 213 K in CDCl₃ a fast conformational movement is already taking place between two identical structures of the complex. It is not possible to determine whether in this complex intermolecular exchange processes are also taking place.The reactions of the anionic [pyrrole-2-CHNR′]^? ligands [(N′N)^?] (R′ = t-Bu, i-Pr, (S)-CHMePh or 2,6-xylyl) with Zn(II) yield the neutral Zn(N′N)₂ complexes. These complexes were synthesized to study the coordination properties of the [pyrrole-2-CHNR′]^? moieties with Zn(II). A ¹H NMR study established that the zinc centres in the complexes containing the prochiral i-Pr or chiral (S)-CHMePh substituents have tetrahedral geometries with Δ or Λ configurations in CDCl₃ at 213 K. These complexes undergo an intramolecular exchange process at higher temperatures (above 260 K when R′ = i-Pr) which involves inversion of the configuration of the zinc centre. A mechanism for this exchange process is proposed.     

Charge modification of the endothelial surface layer modulates the permeability barrier of isolated rat mesenteric small arteries

We hypothesized that modulation of the effective charge density of the endothelial surface layer (ESL) results in altered arterial barrier properties to transport of anionic solutes. Rat mesenteric small arteries (diameter approximately 190 microm) were isolated, cannulated, perfused, and superfused with MOPS-buffered physiological salt solutions. MOPS-solutions were of normal ionic strength (162 mM, MOPS), low ionic strength (81 mM, LO-MOPS), or high ionic strength (323 mM, HI-MOPS), to modulate ESL charge density (normal, high, or low ESL charge, respectively). Osmolarity of MOPS, LO-MOPS, and HI-MOPS was kept constant at 297 mosmol/l, using additional glucose when necessary. Perfusate solutions were supplemented with 1% BSA. Arteries were cannulated with a double-barreled theta-pipet on the inlet side and a regular pipet on the outlet side. After infusion of FITC-labeled dextran of 50 kDa (FITC-Delta50) and the endothelial membrane dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, the dynamics of arterial dye filling were determined with confocal microscopy. ESL thickness, as determined from the initial exclusion zone for FITC-Delta50 on the luminal endothelial surface, was 6.3 +/- 1.4 microm for LO-MOPS, 2.7 +/- 1.0 microm for MOPS, and 1.1 +/- 1.3 microm for HI-MOPS. At low ionic strength, FITC-Delta50 permeated into the ESL with a total ESL permeation time (tauESL) of 26 min, and at normal ionic strength with a tauESL of 20 min. No apparent exclusion of FITC-Delta50 from the ESL could be observed at high ionic strength. In conclusion, we demonstrate that the modulation of solvent ionic strength influences the thickness and barrier properties of the ESL.     

PIP2 signaling in lipid domains: a critical re-evaluation

Microdomains such as rafts are considered as scaffolds for phosphatidylinositol (4,5) bisphosphate (PIP2) signaling, enabling PIP2 to selectively regulate different processes in the cell. Enrichment of PIP2 in microdomains was based on cholesterol-depletion and detergent-extraction studies. Here we show that two distinct phospholipase C-coupled receptors (those for neurokinin A and endothelin) share the same, homogeneously distributed PIP2 pool at the plasma membrane, even though the neurokinin A receptor is localized to microdomains and is cholesterol dependent in its PIP2 signaling whereas the endothelin receptor is not. Our experiments further indicate that detergent treatment causes PIP2 clustering and that cholesterol depletion interferes with basal, ligand-independent recycling of the neurokinin A receptor, thereby providing alternative explanations for the enrichment of PIP2 in detergent-insoluble membrane fractions and for the cholesterol dependency of PIP2 breakdown, respectively.     

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Inherited disorders of HDL metabolism and atherosclerosis

PURPOSE OF REVIEW: Genetic disorders of HDL metabolism are rare and, as a result, the assessment of atherosclerosis risk in individuals suffering from these disorders has been difficult. Ultrasound imaging of carotid arteries has provided a tool to assess the risk in hereditary hypo and hyperalphalipoproteinemia. This review gives a comprehensive summary. RECENT FINDINGS: Epidemiological studies have unequivocally shown that HDL cholesterol levels are inversely related to coronary artery disease risk, but the literature concerning genetic disorders of HDL metabolism provides less convincing information. Fortuitously, we were able to directly compare carotid intima media thickness data of substantial numbers of individuals with mutations in either apolipoprotein A-I (apoA-I), ATP binding cassette AI (ABCA1), lecithin: cholesterol acyltransferase (LCAT) or cholesteryl ester transfer protein. These data show that carriers of an apoA-I mutation exhibit the most pronounced accelerated atherosclerosis compared with those carrying mutations in ABCA1 and LCAT. Heterozygosity for a non-sense mutation in cholesteryl ester transfer protein did, by contrast, not distinguish carriers from controls in terms of intima media thickness progression. We will discuss these results in the context of the current literature. SUMMARY: Intima media thickness studies have provided evidence that hypoalphalipoproteinemia due to mutations in apoA-I, ABCA1, and LCAT is associated with increased progression of atherosclerosis. In contrast, hyperalphalipoproteinemia as a result of loss of cholesteryl ester transfer protein function is associated with unaltered atherosclerosis progression compared with family controls. This insight is of interest, since it can assist in the prioritizing of antiatherogenic therapy by increasing HDL cholesterol levels.     

The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.     

Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission

Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized. It is shown that this FRET pair is best excited at 430 nm. We identify major sources of error and variability in confocal FRET acquisition including chromatic aberrations and instability of the excitation sources. We demonstrate that a novel correction algorithm that employs online corrective measurements yields reliable estimates of FRET efficiency, and it is also shown how the effect of other error sources can be minimized.