The Proinflammatory Environment in Potential Heart and Lung Donors: Prevalence and Impact of Donor Management and Hormonal Therapy

BACKGROUND: Brain stem death can elicit a potentially manipulable cardiotoxic proinflammatory cytokine response. We investigated the prevalence of this response, the impact of donor management with tri-iodothyronine (T3) and methylprednisolone (MP) administration, and the relationship of biomarkers to organ function and transplant suitability. METHODS: In a prospective randomized double-blinded factorially designed study of T3 and MP therapy, we measured serum levels of interleukin-1 and -6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein, and procalcitonin (PCT) levels in 79 potential heart or lung donors. Measurements were performed before and after 4 hr of algorithm-based donor management to optimize cardiorespiratory function and +/-hormone treatment. Donors were assigned to receive T3, MP, both drugs, or placebo. RESULTS: Initial IL-1 was elevated in 16% donors, IL-6 in 100%, TNF-alpha in 28%, CRP in 98%, and PCT in 87%. Overall biomarker concentrations did not change between initial and later measurements and neither T3 nor MP effected any change. Both PCT (P =0.02) and TNF-alpha (P =0.044) levels were higher in donor hearts with marginal hemodynamics at initial assessment. Higher PCT levels were related to worse cardiac index and right and left ventricular ejection fractions and a PCT level more than 2 ng x mL(-1) may attenuate any improvement in cardiac index gained by donor management. No differences were observed between initially marginal and nonmarginal donor lungs. A PCT level less than or equal to 2 ng x mL(-1) but not other biomarkers predicted transplant suitability following management. CONCLUSIONS: There is high prevalence of a proinflammatory environment in the organ donor that is not affected by tri-iodothyronine or MP therapy. High PCT and TNF-alpha levels are associated with donor heart dysfunction. (C) 2009 Lippincott Williams & Wilkins, Inc.

Effect of disability glare on visual performance

The object of the study was to investigate, establish and quantify the relationship between contrast sensitivity, intraocular light scatter and glare. The aim was to establish the effects on vision, in an effort to provide a more comprehensive understanding of the visual world of subjects prone to increased light scatter in the eye. Disability glare refers to the reduction in visual performance produced by a glare source. The reduction in visual performance can be explained by intraocular scattered light producing a veiling luminance which is superimposed upon the retinal image. This veiling luminance lowers contrast thus sensitivity to the stimulus declines. The effect of glare of luminance and colour contrast sensitivity for young and elderly subjects was examined. For both age groups, disability glare was greatest for the red-green stimulus and least for the blue-yellow. The precise effect of a glare source on colour discrimination depends upon the interaction between the chromaticity of the glare source and that of the stimulus. The effect of a long wavelength pass (red) and a short wavelength pass filter (blue) on disability glare was examined. Disability glare was not significantly different with the red and blue filters, even in the presence of wavelength dependent scatter. An equation was derived which allowed an intrinsic Light Scatter Factor (LSF) to be determined for any given glare angle (Paulsson and Sjöstrand, 1980). Corrections to the formula to account for factors such as pupil size changes are unnecessary. The results confirm the suitability of measuring the LSF using contrast threshold with and without glare, provided that appropriate methods are used. Using this formula an investigation into the amount of wavelength dependent scatter indicated that wavelength dependent scatter in normal young, elderly or cataractous eyes is of little or no significance. Finally, it seemed desirable to investigate the effect ultraviolet (UV) radiation has on intraocular light scatter and subsequently visual performance. Overall the results indicated that the presence or absence of UV radiation has relatively little effect on visual function for the young, elderly or cataract patient.

The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells:CD14 and rapid tethering of apoptotic cells

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material. © 2013 Thomas et al.

The value of magnetoencephalography to guide electrode implantation in epilepsy

To investigate if Magnetoencephalography (MEG) can add non-redundant information to guide implantation sites for intracranial recordings (IR). The contribution of MEG to intracranial recording planning was evaluated in 12 consecutive patients assessed pre-surgically with MEG followed by IR. Primary outcome measures were the identification of focal seizure onset in IR and favorable surgical outcome. Outcome measures were compared to those of 12 patients matched for implantation type in whom non-invasive pre-surgical assessment suggested clear hypotheses for implantation (non-MEG group). In the MEG group, non-invasive assessment without MEG was inconclusive, and MEG was then used to further help identify implantation sites. In all MEG patients, at least one virtual MEG electrode generated suitable hypotheses for the location of implantations. No differences in outcome measures were found between non-MEG and MEG groups. Although the MEG group included more complex patients, it showed similar percentage of successful implantations as the non-MEG group. This suggests that MEG can contribute to identify implantation sites where standard methods failed. © 2013 Springer Science+Business Media New York.

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

An X-ray micro-fluorescence study to investigate the distribution of Al, Si, P and Ca ions in the surrounding soft tissue after implantation of a calcium phosphate-mullite ceramic composite in a rabbit animal model

Synthetic calcium phosphates, despite their bioactivity, are brittle. Calcium phosphate-mullite composites have been suggested as potential dental and bone replacement materials which exhibit increased toughness. Aluminium, present in mullite, has however been linked to bone demineralisation and neurotoxicity: it is therefore important to characterise the materials fully in order to understand their in vivo behaviour. The present work reports the compositional mapping of the interfacial region of a calcium phosphate-20 wt% mullite biocomposite/soft tissue interface, obtained from the samples implanted into the long bones of healthy rabbits according to standard protocols (ISO-10993) for up to 12 weeks. X-ray micro-fluorescence was used to map simultaneously the distribution of Al, P, Si and Ca across the ceramic-soft tissue interface. A well defined and sharp interface region was present between the ceramic and the surrounding soft tissue for each time period examined. The concentration of Al in the surrounding tissue was found to fall by two orders of magnitude, to the background level, within similar to 35 mu m of the implanted ceramic.

The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment

The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508

Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [3H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.