To reach and grasp an object in space on the basis of its image cast on the retina requires different coordinate transformations that take into account gaze and limb positioning. Eye position in the orbit influences the image's conversion from retinotopic (eye-centered) coordinates to an egocentric frame necessary for guiding action. Neuroimaging studies have revealed eye position-dependent activity in extrastriate visual, parietal and frontal areas that is along the visuo-motor pathway. At the earliest vision stage, the role of the primary visual area (V1) in this process remains unclear. We used an experimental design based on pattern-onset visual evoked potentials (VEP) recordings to study the effect of eye position on V1 activity in humans.We showed that the amplitude of the initial C1 component of VEP, acknowledged to originate in V1, was modulated by the eye position. We also established that putative spontaneous small saccades related to eccentric fixation, as well as retinal disparity cannot explain the effects of changing C1 amplitude of VEP in the present study.The present modulation of the early component of VEP suggests an eye position-dependent activity of the human primary visual area. Our findings also evidence that cortical processes combine information about the position of the stimulus on the retinae with information about the location of the eyes in their orbit as early as the stage of primary visual area. In humans, goal-directed movements to an object in space are improved by foveal vision, namely by gaze anchoring on the object . Since mvs. lower visual field stimulation [for review see [The C1 component reverses classically in polarity for upper view see ]. ConseqOnce the C1 component was characterized for both upper and lower quadrant visual fields, the effect of eye position on C1 amplitude was measured only for both left and right lower quadrants visual field that is for the most salient C1 component which we observed.Mean peak latencies of C1 was calculated in each subject in response to both left and right lower visual quadrants and for both IN3 and IN4 recording sites. They ranged between 94.8 ms and 98.0 ms that are consistent with the C1 latency range previously observed in numerous studies ,15. For We observed that checkerboard presented in the lower visual field elicited VEPs with modulated amplitudes of the C1 component in respect of eye position. Grand averaged VEPs over the 20 subjects in response to flashed stimuli in the right lower quadrant of the visual field for three different eye positions (0° and 20° both left- and rightward) are shown in Figure In other words, the eye position effect was observed at the occipital recording site contralateral to the direction of the eye deviation. Equivalent VEPs but reverse eye-position effects were obtained in response to left field stimuli. A one-way Friedman repeated measures analysis of variance was conducted for each recording site and for each visual stimulation revealing a significant main effect of eye position on the amplitude of the C1 component. Complete data obtained at both recording sites, for each lower visual field are shown in Figure The overall result of the present study revealed that the amplitude of first major component of VEP elicited by checkerboard (C1) is modulated by the eye position. The previous data linking the C1 component to a striate cortex generator, namely V1 activity (see introduction), led us to suggest that eye position influences the earliest cortical stage of visual processing.One may first suggest that the present results may be explained by the difficulty of maintaining eccentric fixation, which may have altered the pattern of fixational eye movements, such as microsaccades [fast, conjugate jerks, smaller than 1/3°, see ]. RecentThe question also arises if the effects of changing C1 amplitude may be due to oculomotor signals and/or retinal disparity that is to the difference in the position of the visual stimulus on each retina related to relative monitor distance. The absence of any parametric effect for the C1 amplitude between 10° and 20° of eye eccentricity may indicate that eye position effects are not due to the difference in the horizontal retinal disparity, but one may argue that a putative relationship between the horizontal disparity and the C1 amplitude is not linear. We evaluated the difference of both horizontal and vertical disparity between the different eye's deviations in our study  from extrastriate and/or oculomotor structures while the initial C1 sensory response (50–90 ms) was not modulated by attention -15,19-22Finally, Trotter et al. (1999) have shown that an eye position signal  is involved in the neural modulation process dealing with the eye position-dependent visual response observed in area V1 of behaving monkeys. Oculomotor signals coming from extraocular muscle afferents and/or corollary discharges are considered as the substrate of such an eye position signal and have been previously described in V1 ,24.The present results and previous works obtained from neural recordings in monkeys indicate that changes in eye position can modify response properties in V1 that is at the earliest cortical stage of visual processing. Among the visuo-motor processing allowing accurate reaching and grasping movements in space on the basis of the image seen by the retina, the primary visual area may be therefore one of the first cortical relay to convert the image in eye-centered coordinates into a target location by taking into account the position of the eyes in the orbit. It endorses some recent arguments pointing out that V1 could no longer be considered only in relation to the pattern of light falling on the retina but appears to be a cortical area in which contextual influences take place too .Twenty right-handed healthy volunteers with normal visual acuity  participated in VEP recordings after they provided their written informed consent. The study was approved by the Basse-Normandie ethics committee (Caen, France).-1 of spatial frequency) flashed against a black background (ISI = 500–1000 ms) and delivered by a visual stimulator (Nicolet, Madison, USA). The subjects were instructed to fixate continuously the fixation dot and to keep their attention on it, for each quadrant of the visual field so that the projection of the stimulus on the retina was equivalent whatever was the deviated eye position. The edges of the monitor and the space up to 1 meter around the monitor were masked with an opaque black sheet preventing any cue perception in the room except the flashing checkerboard.The subjects were seated on a swivel armchair with their head stabilized with a headrest. An experimenter slowly rotated the armchair and locked it in one of the five different angles: 0°, 10° and 20° both left- and rightward from the center of the monitor leading to five different eye positions Figure . The verWith respect to the purpose of the present study, we recorded VEP from the scalp using the two occipital intermediate sites (IN3 and IN4) of the modified 10–20-system montage  with botWe evaluated the difference of disparity between the different eye's deviations Figure . The difUsing the dot product, for the left eye, α can be expressed as following:a1e1.a1e2 = |a1e1| |a1e2| cos(α)     (1)x,y,z) centred on the cyclopean axis (c):Expressing of the coordinates of the vectors a1e1 and a1e2 in the canonical reference (x a1e2x + a1e1y a1e2y + a1e1z a1e2z     (2)a1e1.a1e2 = a1e1and combining both (1) and (2) led to:x+a/2 cos(θ)) + (d +a/2sin(θ))2 / |a1e1| |a1e2|     (3)cos(α) = (a/2 cos(θ))(e2Similarly, for the right eye, we obtained:x-a/2cos(θ)) + (d -a/2sin(θ))2 / |a1e1| |a1e2|     (4)cos (β) = (-a/2cos(θ)), and combining both (3) and (4) allows to estimate the magnitude of the disparity (η = β-α) in function of the distance from the fixation point for each given eye's deviation. Therefore, we plotted the magnitude of disparity with each distance (e) and for each eye's deviation [-20°, -10°, 10°, 20°] normalized by the disparity calculated for the central eye position (θ = 0°, Figure C1 : early component of visual evoked potentialfMRI : functional magnetic resonance imagingIN3 and IN4 : occipital intermediate recording sitesISI : inter-stimulus intervalV1 : primary visual areaVEP : visual evoked potentialAll authors designed the study. FA and OE carried out the experiments and generated the method for data analysis. OE performed the statistical analysis and generated the method for estimation of the magnitude of disparity. FA and LP wrote the first versions of the manuscript. All authors read, discussed and approved the final version of the manuscript.


Zierath and Hawley discuss how different fiber types affect muscle metabolism and what the signals are that regulate muscle phenotype Skeletal muscle demonstrates a remarkable plasticity, adapting to a variety of external stimuli , includiIn most mammals, skeletal muscle comprises about 55% of individual body mass and plays vital roles in locomotion, heat production during periods of cold stress, and overall metabolism . Thus, ka, and the more anaerobic (glycolytic) fiber is termed FTb. Under aerobic conditions (sufficient oxygen supply to the working muscles), energy is produced without the production of lactate. Under anaerobic conditions (insufficient oxygen supply to the working muscles), energy is produced via the glycolytic pathway, which results in lactate accumulation and in turn limits anaerobic exercise. Thus, muscle fibers can be classified in terms of contractile and metabolic properties , is typically around 55%, with FTa fibers being twice as common as FTb fibers  of 14 elite male long distance runners, 18 good (but not world-class) male long distance runners, and 19 untrained men. The elite group included Olympic medal winners (p < 0.05). The values found for several of the elite runners were the highest observed in human muscle (> 92% ST). Moreover, the ST fibers from the elite runners were 29% larger than FT fibers (p < 0.05), and both ST and FT fibers were larger in the good runners than in the untrained men. Because of the marked hypertrophy (bulk increase) of the ST fibers in the elite runners, the cross-sectional area composed of these fibers was greater than either the good runners or the untrained subjects . When the data from the elite and good runners was combined, a positive correlation between the proportion of ST fibers and the best 6-mile performance time was noted (r = −0.62, p < 0.05).In this regard, the results of  winners  and Amer2 uptake . Indeed, while an athlete's muscle fiber type is an important morphological component and is related to several contractile and metabolic properties  are more likely to determine the upper limits of endurance capacity  had 50% versus 98% ST muscle fibers. Subsequent work  revealedties see , other pcapacity .The close coupling between muscle fiber type and associated morphological, metabolic, and functional properties is not confined to athletic ability. Insulin sensitivity also correlates with the proportion of ST oxidative fibers . Specifi2+/calmodulin-activated phosphatase implicated in nerve activity-dependent fiber-type specification in skeletal muscle, directly controls the phosphorylation state of the transcription factor NFAT, allowing for its translocation to the nucleus and leading to the activation of slow-type muscle proteins in cooperation with myocyte enhancer factor 2 (MEF2) proteins and other regulatory proteins (2+/calmodulin kinase activity is also upregulated by slow motor neuron activity, possibly because it amplifies the slow-type calcineurin-generated responses by promoting MEF2 transactivator functions and enhancing oxidative capacity through stimulation of mitochondrial biogenesis (Skeletal muscle fiber-type phenotype is regulated by several independent signaling pathways . These iproteins . Calciumogenesis .PLoS Biology (PGC1-α, a transcriptional coactivator of nuclear receptors important to the regulation of a number of mitochondrial genes involved in oxidative metabolism, directly interacts with MEF2 to synergistically activate selective ST muscle genes and also serves as a target for calcineurin signaling . New dat Biology  reveals PLoS Biology (The transition from aerobic to anaerobic metabolism during intense work requires that several systems are rapidly activated to ensure a constant supply of ATP for the working muscles. These include a switch from fat-based to carbohydrate-based fuels, a redistribution of blood flow from nonworking to exercising muscles, and the removal of several of the byproducts of anaerobic metabolism, such as carbon dioxide and lactic acid. Some of these responses are governed by transcriptional control of the FT glycolytic phenotype. For example, skeletal muscle reprogramming from a ST glycolytic phenotype to a FT glycolytic phenotype involves the Six1/Eya1 complex, composed of members of the Six protein family . Moreove Biology , a key rWith the 2004 Olympics still fresh on our minds, many will ask: Who has the right stuff to go the distance? Athletes like Olympic champion Frank Shorter are clearly exceptional and represent an extreme in human skeletal muscle phenotype. Realistically, few of us can ever hope to run a marathon in world-class time. However, there may be cause for some optimism for the average mortal, since endurance exercise training in healthy humans leads to fiber-type specific increases in the abundance of PGC-1 and PPAR-α protein in skeletal muscle . Moreove


Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses.Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity.The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting. Acute respiratory tract infections are major causes of morbidity and mortality. In 2000, lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years . The comAs part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. Historically and indeed currently immunofluorescence  and viruIncreasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised -10. HoweOne such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. The ABI PRISM 7000 real-time facility from Applied Biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. However this approach is compromised by the inability of software to allow for target heterogeneity. In addition it does not allow users to adopt clinically validated primer sets from the literature.To address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. Touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming . A detai3 copies per ml for human rhinovirus type 1b to 4.2 × 103 copies/ml for RSV-A. Specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm [Before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. The complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. Classically this approach works well where a single target is under investigation . Howeverlgorithm , were relgorithm . In addiTo test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. Although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and CoV-229E, these were included on the strip for clinical reasons during the period of the study. These findings and their implications are reported.A total of 99 viruses were detected in 84/222 specimens from a total of 79/183 patients and 4/5 National External Quality Assurance Scheme (NEQAS) controls; immunofluorescence did not detect the parainfluenza virus type 2 virus in one of the NEQAS specimens. Viruses were detected in all of the specimen types processed. The molecular strip detected virus in: 16/36 (44.4%) broncho-alveolar lavages, 62/120 (51.6%) nasopharyngeal secretions, 11/35 (31.4%) sputa and 10/31 (32.2%) combined throat and nasal swabs. Immunofluorescence detected virus in: 6/36 (16.6%) broncho-alveolar lavages, 23/120 (19.1%) nasopharyngeal secretions, 1/35 (2.8%) sputa and 1/31 (3.2%) combined throat and nasal swabs.The median age of male and female patients where virus was detected was 3 y (range 2 weeks – 79 years) and 4 y (5 weeks – 81 years) respectively. Sixteen viruses were detected in 14/27 (51.8%) specimens, confirming a respiratory virus in 12 out of 24 (50%) patients investigated in general practice. Seventy-nine viruses were detected in 70/191 (36.6%) specimens, confirming a respiratory virus in 67 out of 159 (42.1%) patients investigated in hospital. Of the 16 viruses detected in specimens from the community, PCR detected all 16 in contrast to a single identification, influenza A (H3), by immunofluorescence.PCR identified one or more viruses in specimens from 84 of the 183 patients and the 4 NEQAS positive specimens, detecting a total of 99 viruses as shown in Table Dual infections were detected in 11/79 (13.9%) patients. The dual infections were: influenza A (H3) and adenovirus (4); influenza A (H3) virus and rhinovirus (2); influenza A (H1) and adenovirus (1); adenovirus and rhinovirus (3); respiratory syncytial virus type B and rhinovirus (1).Nine patients had more than one specimen taken on the same day in which a virus was detected in at least one specimen by PCR. For 5 of the patients the same virus was detected in each of the 2 specimens. The viruses identified were rhinovirus (3), adenovirus (1) and parainfluenza type 3 (1); the latter was also immunofluorescence positive. In 2 cases a rhinovirus was detected in only one of the specimens. As part of a separate rhinovirus validation protocol one of these specimens was subjected to retesting coupled with limited sequencing of the 5' non-coding region amplicon which confirmed the presence of a rhinovirus sequence. Additionally, premature twin boys admitted to intensive care on the same day with severe bronchiolitis, both had evidence of acute rhinovirus infection by PCR. Limited sequencing of the 5' non-coding region of these viruses as part of the rhinovirus validation protocol confirmed the presence of an identical sequence of rhinovirus in both specimens.Immunofluorescence identified a virus in specimens from 28 of the 183 patients and 3/4 NEQAS positive specimens (16.4%), detecting a total of 31 viruses as shown in Table Although touchdown PCR has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets -22, its In this study the large number of targets is the main problem encountered. Using an empirical approach a series of amplification steps linked to a stepped reduction in annealing temperature from 55°C to 46°C was constructed. This proved extremely resilient when used with a wide range of primer sets and included the apparent anomaly of putting adenovirus through an initial reverse transcription step to stream line all of the targets on to a single strip; we have previously reported this approach for testing group F adenovirus alongside norovirus, astrovirus and rotavirus . The touWhere multiple viral targets are sought in clinical practice, we believe that it is only feasible to correlate the performance of the new assay in a head-to-head comparison with that already in routine use. Unfortunately for many clinical laboratories there is an elusion of testing for a wider range of viruses than is the case, by the inoculation of cell lines with a theoretical ability to grow the respective viruses. The annual reports of most clinical laboratories of one of the commonest human respiratory viruses, human rhinovirus, is an example of this; using the touchdown protocol we now report approximately 450 HRV infections per annum. The under reporting of adenovirus by standard methods  and the 3 and 2 × 103 copies per ml) with a generic matrix set (copy number sensitivity of 1 × 103 copies per ml).The ability to simultaneously validate the performance of multiple molecular primer sets in a routine clinical setting is a major accomplishment of the current methodological approach. The results demonstrated that a range of primers from both the medical literature and from in-house development could be amplified with a single generic touchdown protocol. It therefore confirmed the feasibility of directly incorporating primer sets into a standard operating procedure without the necessity for the individual optimisation of cycling parameters. As such the study results should facilitate primer selection and formal critical evaluation as here described. As an example of this enhanced flexibility we have recently replaced the primer sets for influenza A H1 and H3  cross-contamination during manual extraction and (b) contamination of second round adjacent wells with product from a first round positive amplification. The use of the QIAGEN BioRobot for the extraction of all specimens reduced the former while the nature of the strip prevents the latter, since all the wells have separate mixes Table . With boThe results obtained were very encouraging. Although the strip was constructed to detect a wider range of viruses than immunofluorescence, over the period of validation it almost doubled (59 versus 31) the number of viruses that could have been detected by immunofluorescence, including a positive NEQAS specimen which was missed by immunofluorescence. Of this group of viruses the detection of adenovirus showed the most dramatic increase, an observation we have also previously made in a separate study  and whicAs mentioned in the introduction a factor which often impacts negatively on a laboratory's decision to use molecular diagnostics is one of cost. It is worth considering that no matter which assay is chosen for use, it will attract the same overheads needed to provide the infrastructure of a laboratory set-up i.e. building, utilities, staff and equipment. In this regard there are no cheap tests and to use reagent costs as the sole factor in determining which assay to use is somewhat perverse. While the reagent costs of the strip are higher that commercial immunofluorescence reagents by a factor of 3, including extraction, this would undoubtedly narrow if immunofluorescence were capable of closing the pathogen gaps that currently exist e.g. HRV, hMPV. Currently using this approach, we have been able to replace both immunofluorescence and viral culture and this ability makes molecular diagnostics a more cost effective method for diagnosing viral infections. Taking into account the superior range, sensitivity, ability to quantify and speed of molecular techniques it is incredible how little they are used in routine laboratories. With the advent of SARS and the threat of avian influenza, this deficit is now beginning to disturb health care planners at the highest level.Because specimen sampling was not contiguous seasonal peaks were not detected, accounting for the small numbers of respiratory syncytial virus detected and the lack of detection of human metapneumoviruses, parainfluenza virus type 1 and coronavirus 229E; subsequent (unpublished) data from the routine use of the molecular strip support an important role for human metapneumovirus in acute respiratory infections and the sporadic nature of infections caused by parainfluenza type 1 and coronavirus 229E.Several interesting observations need highlighting. First, for immunofluorescence to perform reliably it was essential that a good nasopharyngeal specimen was available. The use of throat and/or nasal swabs with immunofluorescence alone is inappropriate. Second, immunofluorescence was very poor at detecting viruses from patients in the community, again almost certainly because of the universal use of swabs in that setting. Third, the rapid results of immunofluorescence were complemented by the touchdown protocol which can report definitive results within 24 hours, considerably faster than culture. Fourth, the molecular strip was better at detecting multiple infections. Even allowing for the inability of immunofluorescence to detect rhinoviruses, it should have detected the mixed adenovirus and influenza virus infections. Although immunofluorescence is capable of diagnosing dual infections, its routine use along with culture probably grossly underestimates their prevalence. The most plausible explanation is that the molecular technique detects infections where one of the viruses is below the detection threshold of immunofluorescence. These low level viruses are either just starting or more likely reaching the end of an infectious episode (latency is less likely) and this raises the previously unaddressed question of their role in viral respiratory pathogenesis. Fifth, the extent of rhinovirus infections was very significant. Their clinical significance ranged from acting as a definitive respiratory pathogen to a less certain role when acting as the most frequently detected co-pathogen in mixed infections.In conclusion the use of the touchdown protocol with pre-dispensed and quality checked primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses for immunofluorescence negative specimens. Immunofluorescence results were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol is a convenient and reliable method of testing for multiple viruses in a routine setting. Its generic nature makes it especially useful for introducing test repertoire modifications e.g. incorporating primers for the newly identified coronaviruses SARS-CoV and HCoV-NL63.A total of 222 specimens were included in the validation between January 2002 and June 2003, including 14 from an influenza surveillance scheme. The specimens were collected from 183 patients including: 103 male, median age 3.5 y (7 m – 84 y); 80 female patients, median age 9 y (7 m – 84 y); both male and female ages were skewed towards the lower age ranges, and 5 national external quality assurance scheme (NEQAS) specimens (4 positive, 1 negative). One hundred and fifty-nine patients were in hospital and 24 were in the community at the time of sampling. Specimens tested consisted of a wide range of specimens including: broncho-alveolar lavage (36), nasopharyngeal secretions (120), sputum (35) and combined throat and nasal swabs (31).Nasopharyngeal secretions, broncho-alveolar lavage and sputum specimens were received in dry sterile containers at ambient temperature. Upon receipt they were re-suspended in 2 ml of virus transport medium (VTM) consisting of phosphate buffered saline pH 7.1, bovine serum albumin 7.5 μg/ml, penicillin G sodium 1000 units/ml, streptomycin sulphate 1000 μg/ml and amphotericin B 2.5 μg/ml. Throat and nasal swabs were received in 2 ml of VTM and vortexed on arrival to release cells attached to the fibres of the swab. An aliquot of 410 μl was taken off for extraction after which the specimens were centrifuged at 2600 g for 5 min and the resulting cell deposits air-dried on glass multi-well slides and fixed in acetone prior to testing. Immunofluorescence was set up on the respiratory specimens using commercial reagents according to the manufacturer's instructions, and was able to detect: influenza A, influenza B, respiratory syncytial virus, adenovirus, parainfluenza type 1, parainfluenza type 2 and parainfluenza type 3 (Dako diagnostics, Ely, UK).A volume of 200 μl of the respiratory specimen suspension was extracted on a QIAGEN BioRobot 9604 using the Blood and Body Fluid Vacuum Protocol of the QIAamp DNA Blood Kit (Qiagen Ltd., Crawley, England, U.K). This protocol allows the co-extraction of both RNA and DNA simultaneously.Simultaneous amplification of all targets was facilitated by using a standard 8 well multi-well PCR strip to which all mixes were pre-dispensed and stored frozen; this format is referred to in the paper as the "respiratory strip" because of the respiratory nature of the targets. The respiratory strip targeted the following 12 common respiratory viruses: influenza A H3), influenza A (H1), influenza B, respiratory syncytial virus type A, respiratory syncytial virus type B, adenovirus, coronavirus 229E, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus and human metapneumovirus. The final configuration of the single and multiplex primer mixes in the 8 well strip are shown in Table , influenEach primer master-mix was made-up and titrated against a known positive control before being aliquoted and dispensed into its respective well of the 8-well microtube strip. The strips were stored frozen at -20°C until used. A positive control was also aliquoted and stored separately at -20°C until used. For the duration of the study the positive control was the cloned target of parainfluenza virus type 1; a negative control was not deemed necessary.4, 1 unit AMV reverse transcriptase, 1 unit Tfl DNA polymerase, 0.2 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP) and 1 μM outer primers.First round volumes were made-up in Access RT-PCR buffer (Promega, Southampton, England, U.K) and in the final 10 μl volume contained the following reagent amounts: 1.5 mM MgSO2, 50 mM KCl, 0.1% Triton X-100, 0.2 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP), 0.25 units of Taq DNA polymerase (Promega) and 0.2 μM inner primers.Second round volumes were made-up in Taq Buffer B (Promega) and in the final 10 μl volume contained the following final amounts: 10 mM Tris-HCl (pH 9.0), 3.5 mM MgClFirst round amplification was performed on 2 μl of extract added to 8 μl of first round primer master-mix per well. Second round amplification was performed on 0.2 μl of the first round reaction added to 9.8 μl of second round primer master-mix per well; a multi-channel pipette facilitated the transfer of the 8 volumes in one step. The positive control was run on the eighth well of each strip. The second round products were run on ethidium bromide stained 2% agarose gels and photographed. Specimens were reported positive when respectively the correct size bands and the positive control bands were present.Amplification was carried out on a range of thermal cyclers including the Applied Biosystems GeneAmp 2400 and 9700 series and a DNA Engine Tetrad PTC 225 (MJ Research, USA). The first and second round amplification protocols consisted of 36 identical cycles with the exception that (a) a reverse transcription step of 48°C-10 min preceded the first round and (b) a hot-start preceded the second round by transferring the strip directly from ice to a thermal cycler held at 94°C. The touchdown protocol consisted of 6 steps as follows: (a) initial denaturation (94°C-2 min); (b) 3 cycles of denaturation (94°C-30 s), annealing (55°C-30 s) and extension (72°C-30 s); (c) 3 cycles of denaturation (94°C-30 s), annealing (52°C-30 s) and extension (72°C-30 s); (d) 20 cycles of denaturation (94°C-30 s), annealing (49°C-30 s) and extension (72°C-30 s); (e) 10 cycles of denaturation (94°C-30 s), annealing (46°C-30 s) and extension (72°C-30 s); (f) 72°C for 5 mins.PVC: Touchdown and molecular strip design and manuscript preparation.GMO: Early application of touchdown cycling to respiratory samples.HJO'N: Study protocol and manuscript preparation.CMcC: Study protocol and manuscript preparation.DDEO: Routine application of touchdown protocol during study.FM: Routine application of touchdown protocol during study.SJM: Routine application of touchdown protocol during study.SAF: Primer-mastermix manufacture and quality control.DEW: Early application of touchdown cycling to respiratory samples and manuscript preparation.MF: Routine application of immunofluorescence protocol during study.JS: Design and validation of primers for human metapneumonavirus detection.


When James Watson and Francis Crick reported the structure of DNA in 1953, the mechanism of inheritance was instantly apparent. The complementary pairing of the DNA bases in the double helix, the pair famously wrote, “immediately suggests a possible copying mechanism for the genetic material.” The structure helped explain one of the central problems of modern biology: how does genetic material get faithfully replicated and then passed on from generation to generation? It was long thought that DNA is the only unit of inheritance.Since then, it's become clear that molecules of DNA are packaged into highly organized superstructures that themselves are inherited. These structures play a significant role in the regulation of genes by preventing or facilitating protein–DNA interactions. In the eukaryotic cell (a cell with a nucleus), DNA exists as long threadlike molecules—a typical human cell contains some 6.5 feet (2 meters) of DNA—that associate with a variety of proteins to form a network called chromatin. Genomic DNA wraps around specialized DNA-packing proteins called histones to form nucleosomes, which condense chromatin into chromosomes and thereby influence chromosome behavior. Chromosomes are in turn packaged in increasingly higher levels of organization, with some parts being dispersed and others condensed. The most condensed region is called heterochromatin, or silent chromatin. Gene expression is largely silent in these regions, since the proteins required for transcription can't access DNA to transcribe genes when chromosomes are so tightly packed. Other regions of chromosomes exist in an extended state, called euchromatin. This is the most genetically active state; with genes exposed, transcription can easily occur.PLoS Biology, Jasper Rine, Hiten Madhani, and colleagues identify and characterize the function of a protein complex that helps deposit the variant H2A.Z onto euchromatin in yeast.As chromatin shifts between these states, it influences gene expression, largely through the interactions of histones and large protein complexes that together assemble, remodel, and modify chromatin. Since proper cell function depends largely on activating the right gene at the right time, mechanisms have evolved that protect active genes from the intrusions of silencing structures like heterochromatin. Both euchromatin and heterochromatin respond to mechanisms that resist encroachments of the opposite state. One mechanism involves replacing “canonical”  histones with a histone variant. Previous work on yeast from Hiten Madhani and colleagues had shown that one histone variant, called H2A.Z, is found specifically in euchromatin and prevents silent chromatin from spreading into adjacent euchromatic regions. While researchers have characterized some of the mechanisms that deposit canonical histones onto euchromatin, they knew little about the mechanisms that deposit variant histones. In this issue of To investigate which proteins help direct H2A.Z to specific chromosomal locations, the authors isolated H2A.Z, along with whatever proteins were associated with it, from yeast cell extracts. They determined that 15 proteins were true binding partners of H2A.Z and that 13 of them form a complex called SWR1-Com. The largest subunit of this complex, called Swr1p, belongs to a well-known family of adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes (they use the energy of ATP to power remodeling) that provide access to DNA in chromatin. Rine, Madhani, and colleagues show that protein subunits of SWR1-Com associate specifically with the histone variant H2A.Z. By comparing the gene expression profiles of yeast mutants lacking the H2A.Z-encoding gene with mutants lacking the Swr1p-encoding gene, the authors show that H2A.Z depends on the SWR1-Com protein complex to function. Most importantly, they show that SWR1-Com is required in living cells to deposit H2A.Z onto euchromatin. Interestingly, the authors note, SWR1-Com shares subunits with a histone-acetylating enzyme involved in the regulation of transcription  and with another chromatin remodeler, which suggests that biochemical modifications of the subunits on histone “tails” may play a role in replacing H2A with H2A.Z.This histone–protein complex, the authors conclude, represents a chromatin remodeling machine with a novel function, revealing a new role for Swr1p-type enzymes and a novel mechanism of genome regulation. By preventing the spread of silent chromatin into transcriptionally active chromosomal regions—the result of the interaction described here—this mechanism allows the cell's gene expression program to operate with precision and on schedule. Since chromosomes can be inherited by daughter cells in this active state, such mechanisms ensure that gene expression programs essential for ongoing fundamental processes like embryogenesis and cellular differentiation proceed without interference.


DAT1) has been associated with a range of psychiatric phenotypes, most notably attention-deficit hyperactivity disorder. The mechanism for this association is not yet understood, although several lines of evidence implicate variation in gene expression. In this study we have characterised the genomic structure of the 9- and 10-repeat VNTR alleles, and directly examined the role of the polymorphism in mediating gene expression by measuring comparative in vitro cellular expression using a reporter-gene assay system.The 10-repeat allele of a variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region of the dopamine transporter gene (Differences in the sequence of the 9- and 10- repeat alleles were confirmed but no polymorphic differences were observed between individuals. There was no difference in expression of reporter gene constructs containing the two alleles.DAT1 expression and that the associations observed with psychiatric phenotypes may be mediated via linkage disequilibrium with other functional polymorphisms.Our data suggests that this VNTR polymorphism may not have a direct effect on  DAT1) is associated with a range of psychiatric phenotypes. In particular, the 10-repeat allele of this polymorphism has been widely associated with attention deficit hyperactivity disorder (ADHD) (e.g. [The dopamine transporter (DAT) mediates uptake of dopamine into presynaptic neurons, and is a major target for various pharmacologically active stimulants such as cocaine. Genetic association studies provide considerable evidence that a variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region (UTR) of the dopamine transporter gene DAT is assocD) (e.g. ), althouD) (e.g. ). The meD) (e.g. . The VNTD) (e.g. .in vivo studies using single photon emission computed tomography (SPECT) show an increased density of DAT in ADHD probands compared to controls [DAT1 genotype suggests that the 10-repeat allele is associated with a positive response to the drug [DAT1 VNTR on methylphenidate response, although they find poor response is associated with the 10-repeat allele [DAT1 messenger RNA (mRNA) levels in cerebellum, temporal lobe and lymphocytes and observed that increased levels of DAT1 expression were associated with the number of 10-repeat alleles [Several controls -10, althcontrols . In addicontrols ,13]. Furcontrols . Finallythe drug  as wouldt allele ,16. Prev alleles . These dDAT1 VNTR in vitro, although the results from these studies are inconclusive. Michelhaugh et al demonstrated that the human DAT1 9-repeat VNTR enhances transcription in SN4741 cells, obtained from a mouse-embryonic substantia nigra-derived cell line [DAT1 gene. It has been postulated that large repeat motifs in the 3'UTR of genes may alter mRNA stability, but such effects would not be detected in the research presented by Michelhaugh et al. Furthermore, it has been shown that the enhancing effect of sequence elements is largely determined by their relative location [DAT1 [DAT1 VNTR alleles from several primate species transfected into the human neuroblastoma cell-line, SK-N-SH [DAT1 gene expression.A number of groups have investigated the functional role of the ell line . Howeverlocation . Fuke etlocation . They foon [DAT1 . Again,  SK-N-SH . They fo SK-N-SH . They coin vitro cellular expression implementing a reporter-gene assay system in which the VNTR has been inserted in an appropriate 3' configuration, and using human cell-lines known to express DAT1. Analysis was limited specifically to the VNTR sequence itself, so that any functional effect detected could be attributed solely to this candidate polymorphism. Another aim of this study was to fully characterise the genomic structure of the DAT1 VNTR. It is possible that any functional effects of the VNTR are not mediated by repeat length, but instead by variation in the actual sequence of the repeat units. Recent work on the DRD4 exon III VNTR sequence has demonstrated that there can be considerable variation within the sequence of large VNTRs [The aim of this study was to examine directly the role of the VNTR polymorphism in mediating gene expression by measuring comparative ge VNTRs . Such vaDifferences were found between repeat motifs , and nine variants (labelled A-I) were detected in total . Some slight differences were noted between the two cell lines: in both SH-SY5Y cells and HEK-293 cells, the pGL3-basic plasmid, which contains neither promoter nor enhancer sequences, generated the lowest transcriptional activity as expected. In the SH-SY5Y cells, control plasmids containing both an enhancer and promoter showed significantly higher levels of transcriptional activity than the promoter plasmids, although this difference was not seen in the HEK-293 cells.Table DAT1 VNTR; and second to investigate the effect of the 9- and 10-repeat VNTR alleles on levels of transcription. It is possible that any effects, both on behavioural phenotype or levels of DAT1 expression, commonly associated with the VNTR are due to polymorphisms within the repeat rather than the actual length of the VNTR itself. This theory has often been postulated to explain cases of non-replication of association studies with complex disorders. Recent work on the DRD4 exon III VNTR polymorphism has highlighted the existence of both within individual and between individual variation in repeat motif sequence [st and 2nd repeat motifs within a certain length DRD4 VNTR allele may differ in sequence in a single individual, but there is also variation within the 1st motif between different individuals. We sequenced the DAT1 VNTR in individuals homozygous for either the 9- or 10-repeat allele. Differences were found between repeat motifs in both the 9- and 10-repeat alleles, and nine variants were detected in total. Interestingly, although differences were found between the 9- and 10-repeat alleles, the order of the motifs was identical for all examples of each allele size. Given that not one variation in these sequences was observed in the 60 chromosomes sequenced, it can be concluded that any between individual variations that do occur are extremely rare and unlikely to be the cause of common disorders such as ADHD.The aims of this study were twofold: first to characterise the genomic structure of the sequence . In otheDAT1 3'UTR for novel polymorphisms in a Japanese population, but only detected a G>A SNP located upstream of the VNTR at position 2319 [DAT1 VNTR in a small number of Japanese subjects with results identical to those we describe [DraI restriction site, which they claim to be within the 10-repeat allele [Previously, Ueno et al screened the ion 2319 . Fuke etdescribe . Miller t allele . Closer t allele  may be tDAT1 VNTR is perhaps surprising given the number of polymorphisms seen in other large VNTRs . VNTRs Rs (e.g. -29. FurtRs (e.g. ), the avnylation . Alternain vitro possible functional consequences of the DAT1 VNTR polymorphism. We made constructs containing the 9- and 10-repeat alleles of the DAT1 VNTR and the luciferase reporter gene, and transiently transfected them into SH-SY5Y and HEK-293 cells. Constructs containing the VNTR alleles gave slightly lower levels of luciferase activity compared to vectors without the inserts. Although these differences were not statistically significant, they appear to go against the conclusions of a previous study in which the VNTR sequence acted as a strong enhancer of transcription [The second aim of this study was to examine cription . Howevercription . Furthercription . The discription , which dcription  and so icription  is speciDAT1 VNTR alleles. Other transient expression studies that have compared the VNTR alleles have provided mixed results. Fuke et al found that luciferase expression was significantly higher in cells transfected with vectors containing the 10-repeat allele compared to the 7-repeat or 9-repeat alleles [DAT1 [We found no significant differences in luciferase activity between constructs containing the 9- or 10-repeat  alleles . Their ses [DAT1 . Finallyes [DAT1 .not be functional. Unlike the studies of Fuke et al and Miller & Madras [DAT1 gene. Genetic association studies with ADHD, SPECT brain imaging studies, and correlations with levels of DAT protein and DAT1 mRNA all suggest that a variant associated with DAT1 expression is present in this region. Given that in each of these associations, the VNTR has been nominated as the causative polymorphism, it is likely that the real risk variant is in strong LD with it. Ueno et al and Miller et al both report novel SNPs located within the 3'UTR and close to the VNTR [DAT1 and is the real risk variant.Therefore, our data suggest that the VNTR polymorphism itself may & Madras ,21, our the VNTR ,26. It iin vitro studies of gene expression reflect patterns seen in vivo. Future work could employ animal models to characterise more realistically the effect of the VNTR on DAT1 expression. Second, while we ensured that we used cell-lines that naturally express DAT1, and inserted the VNTR into the correct 3'UTR location of the luciferase reporter gene, our constructs could have been improved by using a homologous DAT promoter. Third, we only cloned a very small portion of the DAT1 gene. Even though this was necessary to examine functional effects specific to the VNTR, the fact that the majority of the DAT1 gene was absent means that it is likely that several cis-acting regulatory elements were not present in the constructs and thus the observed expression may not reflect the actual regulation of the gene. Finally these studies have only analysed DAT1 gene expression in the basal state, and complexities such as the induction of expression by factors such as cellular signals would thus be missed. Future work should focus on systematically characterising the remainder of the 3'UTR to discover the functional effects of other polymorphisms in this candidate region.There are several obvious limitations to this study. First, it is not known how well DAT1 VNTR alleles, and directly examined the role of the polymorphism in mediating gene expression by measuring comparative in vitro cellular expression using a reporter-gene assay system. No expression differences were observed between the 9- and 10-repeat alleles suggesting that this polymorphism may not have a direct effect on DAT1 function.In this study we have characterised the genomic structure of the 9- and 10-repeat DAT1 VNTR and performing expression analyses it was imperative to characterise fully its structure. If there was variation within the VNTR between individuals who have repeats of the same length, then these variants could confound expression analyses based simply upon length. Thirty individuals of predominantly Caucasian ethnicity, homozygous for either the 9- or 10-repeat allele of the VNTR, were selected for sequencing. The VNTR region was amplified on an MJ PTC-225 thermal cycler (MJ Research, Massachusetts, USA) using the primers 5'- TGT GGT GTA GGG AAC GGC C-3' and 5'- CAT TCG CAA ACA TAA AAA CTG TTG T-3' using a standard PCR protocol with a proofreading polymerase and an annealing temperature of 58°C. PCR products were run on a 2% agarose gel stained with ethidium bromide, and then purified using the QIAquick Gel Extraction Kit (Qiagen, Crawley, UK). Purified fragments were sequenced using an ABI BigDye Terminator (v3.0) Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA, USA) and electrophoresed on an ABI 3100 Genetic Analyzer (PE Applied Biosystems). Sequencing traces were analysed using Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA) and multiple samples aligned to aid mutation detection.Before cloning the Xba I restriction site (TCTAGA) immediately downstream from the luciferase reporter gene, enabling inserts to be cloned into the 3'UTR. Primers were designed to directly flank the DAT1 VNTR and to both of these an Xba I restriction site, along with three extra bases of DAT1 sequence, were added. The primer sequences used were: 5'-TGT TCT AGA TTG TGG TGT AGG GAA CGG C-3' and 5'-AGG TCT AGA AGA GTG TTG GTC TGC AGG CT-3'. The aim of this project was to concentrate solely on the functional significance of the VNTR, so flanking regions around the VNTR were kept as small as possible. Using these primers, the VNTR was amplified in individuals homozygous for the 9- and 10-repeat alleles using standard PCR conditions with an annealing temperature of 55°C. The PCR products were isolated from a 2% agarose gel as described above. The purified fragments were cloned into pCRII TA cloning vectors (Invitrogen, UK), which were then transformed into TOP10F E. Coli cells (Invitrogen, UK) following the manufacturers protocol. Colonies containing recombinant plasmids were identified using X-gal and IPTG. DNA from colonies containing recombinant plasmids was prepared using a Qiagen Midi-prep kit (Qiagen, UK), and the presence of insertions was verified using Xba I restriction enzyme digestion and further checked using direct plasmid sequencing. The Xba I digested 9- and 10-repeat VNTR inserts were run on a 2% agarose gel and purified as described above. The fragments were cloned into pGL3-Control and pGL3-Promoter vectors. The pGL3-Control vector contains both SV40 promoter and enhancer sequences whereas the pGL3-Promoter vector contains only an SV40 promoter. Additionally the pGL3-Basic vector which lacks eukaryotic promoter and enhancer elements was tested on its own without any insert, for transcriptional activity and served as a negative control. Five colonies from each of the four cloning reactions (pGL3-Control-9rpt, pGL3-Promoter-9rpt, pGL3-Control-10rpt, and pGL3-Promoter-10rpt) were selected. Following plasmid DNA preparation, the presence and orientation of the inserts was again verified by Xba I digestion and fluorescent sequencing. New DNA stocks for each of the 8 plasmids to be used in subsequent transfection experiments (pGL3-Control, pGL3-Promoter, pGL3-Basic, pRLSV40, pGL3-Control-9rpt, pGL3-Promoter-9rpt, pGL3-Control-10rpt, and pGL3-Promoter-10rpt) were prepared, and tested again by Xba I digestion.The pGL3 expression vector family (Promega, UK) contains an in vitro studies of gene expression using reporter genes is that the cell-lines used do not naturally express the gene of interest (i.e. DAT1). Before we transfected our constructs into the SH-SY5Y and HEK-293 cell lines they were tested for DAT1 expression using quantitative RT-PCR. Both cell lines were found to naturally express DAT1 making them suitable for our experiments (data not shown). In addition previous in vitro studies have shown regulation of the DAT1 gene in both the HEK-293 and SH-SY5Y cell lines [SH-SY5Y and HEK-293 cells were purchased from ATCC (VA, USA). The SH-SY5Y cell line is a thrice-cloned subline of the neuroblastoma cell line SK-N-SH. The HEK-293 cell line is derived from human embryonic kidney. A problem with many ll lines ,32, sugg2. Transfection of the SH-SY5Y cells was carried out using a calcium phosphate transfection kit (Invitrogen, UK). The two different transfection methods used were found to be optimal for each line respectively. The HEK-293 cell line was transiently transfected using Lipofectamine 2000 (Invitrogen, UK). Transfection efficiencies were normalised by the co-transfection of the Renilla vector, pRL-SV40 (Promega, UK). Following transfection, the cells were allowed to grow for 48 hours. The cells were then washed and 500 μl cell lysis buffer (Invitrogen, UK) was added to each well. The firefly luciferase and Renilla luciferase assays were carried out using the Dual-Luciferase Assay System (Promega, UK) following the manufacturers protocol. All transfections were done in triplicate, and repeated at least three times. All comparisons between constructs were analysed by one-way analysis of variance (ANOVA), followed by Tukey post-hoc analysis for pairwise comparisons between specific plasmids.Both cell lines were cultured in 6-well tissue culture plates containing Minimun essential medium Eagle (ATCC) supplemented with 10% foetal bovine serum (Invitrogen, UK). Cells were grown at 37°C in a humidified atmosphere containing 5% COJM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. UD participated in the overall design and co-ordination of the study with supervision of cell culture techniques. PA/IC contributed to the interpretation of findings and general points of the experimental design. All authors read and approved the final manuscript


The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha  represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells).Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 . NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells  were measured using commercially available kits.In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells.In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells. Oxidative stress is an important factor of acute lung injury. Prolonged exposure to high concentrations of oxygen (hyperoxia) during mechanical ventilation represents a life-saving intervention for critically ill patients. However, it also induces oxidative stress to the lung. The development of therapeutic strategies, aiming to prevent lung injury depends on a better understanding of the underlying pathways of hyperoxia-induced pulmonary damage.Severe, long lasting hyperoxia causes an inflammatory reaction with an influx of inflammatory cells, cell proliferation and hypertrophy, an increase of cytokines, apoptotic activity and subsequent morphologic evidence of lung injury . The firFollowing the concept that hyperoxic lung injury is a continuous process, we assumed that appropriate metabolic changes of TIIcells start during the initiation phase. This would then, in response to longer lasting severe hyperoxia or an additional stress, merge in apoptosis in the post-initiation phase . FactorsElevation of tumour necrosis factor α (TNFα) represents one of the first pulmonary responses to hyperoxia. Pre-treatment of animals with antibodies directed against TNFα reduces hyperoxia-induced lung injury, strongly suggesting a causal relationship between TNFα and hyperoxic lung . TNFα isIn this paper, we tested the hypothesis that TNFα activates caspases in the initiation phase of pulmonary oxygen toxicity in TIIcells without a significant increase in apoptosis. Since apoptosis is modulated by cell-matrix and cell-cell interactions in cultured TIIcells isolated from animals with acute lung injury ,25. we uad libitum. Preparation of the bronchoalveolar lavage, alveolar macrophages and TIIcells was carried out as previously described [Wistar rats (body wt 120 g), each in an individual plastic chamber were continuously gassed with 100 % oxygen for 48 hours. Water and food was available escribed .Immunohistochemistry and microscopy were carried out as previously described . The folFor double staining, the labelled preparations were analysed using a confocal laser scanning microscope (CLSM, Leica Microsystems AG, Wetzlar, Germany), equipped with an argon/krypton laser. Images were taken using a 40 × NA 1.3 oil objective to fluorescent excitation and emission spectra for Alexa 488 (excitation 490 nm, emission 520 nm) and for Alexa 594 (excitation 541 nm, emission 572 nm). With the dual-channel system of the confocal microscope, dual-emission (535/590 nm) images were recorded simultaneously with a scanning speed at 16 s/frame (512 lines). Images were obtained and processed using TCS NT Version 1.5.451 (Leica Microsystems AG, Wetzlar, Germany). As controls, the tissue slides were incubated with the Alexa-labelled second antibodies only. No unspecific binding of the second antibodies occurred (results not shown).For threefold staining Figure , fixed lLaser scanning confocal microscopy was performed using a ZEISS LSM 510 system with Axiovert microscope (Carl Zeiss Jena GmbH, Jena, Germany) with 40×/1.3 Oil Dic or 63×/1.4 Oil Dic objective, equipped with an argon, helium/neon and violet laser set to 488, 543 and 405 nm, respectively. The multitrack standard FITC/Rhodamine/DAPI configuration was selected.Sections of rat lung were prepared as described . After dCytoplasmic histone-DNA fragments were quantified using the Cell Death Detection ELISA (Roche, Mannheim, Germany).We used the TACS™ Annexin V-FITC Detection kit (R&D Systems, Wiesbaden, Germany) to quantify the population of early and late apoptotic cells in percent of total cells. The tests were performed according to the protocols of the manufactures.6 TIIcells for each group and for each caspase with the Colorimetric assays from R&D Systems Inc. (Wiesbaden, Germany). When the pro-caspases had to be determined, an aliquot of the lysate (corresponding to 2 × 106 cells) was preincubated with 0.1 μg granzyme  for 30 min at 37°C.Activities of caspases 3, 8 and 9 were determined in the lysates of 8 × 10Activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody , that recognises an epitope which includes the nuclear location signal of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-κB. Thus, only the activated form of NF-κB was measured. The semiquantitative estimation of NF-κB subunit by confocal microscopy was carried out as recently described in detail .Translocation of NF-κB to the nucleus was assessed as described by Li et al. by immunoblotting of nuclear extracts using a rabbit polyclonal antibody (biomol GmbH, Hamburg, Germany) directed against the p65-subunit .(EMSA) was employed to detect the activated transcription factor NF-κB. Because this method is based on the binding of the transcription factors to their specific DNA recognition sequences, it is highly specific. Labelling of the NF-κB consensus oligonucleotide and handling of the assay were as described by the manufacturer (Gel Shift Assay Systems, Promega GmbH, Mannheim, Germany). Briefly, 50 micrograms of TIIcell nuclear extract were preincubated in reaction buffer for 10 minutes at RT. A [32P]-labelled oligonucleotide (Promega GmbH, Mannheim, Germany) which contains DNA binding sites for NF-κB transcription factors was then added to the reaction mixture and incubated for 20 minutes at RT. The complexes were separated on a 4% polyacrylamide gel that was dried and exposed to autoradiography. The specificity of the DNA-binding protein for the putative binding site was established by competition experiments using unlabelled NF-κB consensus oligonucleotide.Wistar rats were lightly anaesthetised by inhalation of ether. The rats obtained intratracheally 50 μg goat IgG (PERBIO SCIENCE, Bonn, Germany) or 50 μg goat polyclonal anti-rat TNFα antibody (Santa Cruz Biotechnology, Heidelberg, Germany) per animal, respectively. Thereafter the rats were kept at hyperoxic conditions as described in hyperoxia. After 48 hrs, the TIIcells were isolated and TNFRI expression and caspase-3 activity were determined as described above.For the determination of the TNFα concentration in TIIcells, macrophages, plasma or cell-free bronchoalveolar lavage, we used a commercially available ELISA kit from Biosource (Ratingen, Germany). The determination of different apoptotic markers , WesternDifferences between two groups were assessed using the Student's t-test. Probability values < 0.05 (two-tailed) were considered significant .in vivo; at least not in the initiation phase.To test the general usefulness of our hypothesis, we first characterised the oxygen-induced changes in lung-tissue-sections. Immunohistochemically, we observed a clear increase in TNFα, TNFRI, and caspase 3 activities in lung tissue of hyperoxic rats in relation to normoxic animals in the initiation phase Figure . Albeit in vivo. Following our hypothesis, we examined whether TIIcells undergo oxidative stress at our conditions, and whether an increase of TNFα, TNFRI expression, and caspase-3 activity appears in isolated TIIcells.The results indicate that apoptotic parameters as are TNFα content, TNFRI expression and caspase-3 activity increase in lung tissue during the initiation phase but do not induce TIIcell apoptosis The determination of cellular GSH, oxidised GSH (GSSG), and the activity of the GSH-reductase showed the oxidative burdening of TIIcells. In response to hyperoxia, the GSSG content significantly increased and the GSH reductase activity significantly decreased Table . The GSHThe ratio GSH/(GSH+GSSG) is one of the most sensitive parameters to describe oxidative burdening. Our values are comparable to the values found by van Klaveren et al. in freshly isolated type II cells (0.816 versus 0.815) . This raNF-κB activation has been described as an indicator of oxidative stress ,34. In rThe concentration of TNFα significantly increased in response to hyperoxia of rats in plasma, alveolar fluid, lung macrophages and TIIcells Table  as deterin situ that the activation of caspase 3 actually occurs in TIIcells and that caspase activation as a response to an unspecific stress, e.g. isolation, can be excluded , whereas the expression of Fas, a member of the same receptor family, did not change Figure . TNFRI-mAs shown in Table In order to demonstrate the causality between hyperoxia and TNFRI-mediated activation of caspases, we attempted to bind TNFα by anti-TNFα antibodies. Table The content of individual mRNAs in TIIcells was determined by Real-time PCR. In agreement with microarray analysis in total lung of mice  we foundt of -1.15 indicates a 2.2-fold increase of mRNA content.In response to sublethal hyperoxia the mRNA content of TNFα, TNFRI and caspases increased Table . The incAs mentioned above in this section, no increase of apoptosis was detected in TIIcells in response to sublethal hyperoxia by immunohistochemistry Figure . To confAfter hyperoxia for 48 hrs, we detected an activation of caspase 8 and caspase 3. Much to our surprise, we found no increase in apoptosis of TIIcells although the activity of these caspases increased. However, apoptosis might occur later than 48 hrs and will not necessarily arise concomitantly with caspase activation. Thus, following hyperoxia the animals were kept for 24 and 48 hrs under normoxic conditions to test TIIcells for appearance of apoptosis at a later time point.During normoxia, caspase 3 activity gradually decreases compared to control, whereas apoptosis did not change significantly as detected by cell death detection ELISA and by the number of early and late apoptotic cells Table .Short-time hyperoxia, as used in this experiments represents the initiation phase of lung injury . We starFor the first time, we show that sublethal hyperoxia causes not only an increase in TNFα concentration in lung tissue (as previously published ,39), butIt has been widely assumed that macrophages are the source of TNFα in alveolar fluid . Our finCellular effects of TNFα are mediated mainly by its specific receptor, TNFRI. In agreement with our hypothesis, the expression of TNFRI in TIIcells is up-regulated on mRNA and protein level in response to sublethal hyperoxia, while the Fas-expression did not change, although both receptors belong to the same family Figure . It may The intracellular part of the transmembrane TNFRI protein contains the "death domain" which is responsible for the activation of caspase 8. This can trigger the path to the final part of apoptosis via activation of caspase 3 . In lineTo check whether this pro-apoptotic state in TIIcells is indeed triggered by TNFα and whether it may be reversible, anti-TNFα antibodies were administered intratracheally prior to hyperoxic exposure. We show that this treatment completely prevented hyperoxic-induced increase of TNFRI expression and caspase 3 activation.It is a widely accepted concept that activation of caspase 3 marks the "point of no return" in the pathway of apoptotic death of mammalian cells. However, we found that neither in lung tissue nor in freshly isolated TIIcells apoptosis occurs in response to sublethal hyperoxia despite the significant activation of caspases 8 and 3. In other words, the increase of caspase 8 and 3 in response to sublethal hyperoxia did not mark the "point of no return" in TIIcells. This interpretation of our results is strongly corroborated by Perfettini and Kroemer . They suvia strong mitochondrial damage resulting in cytochrome c release and caspase 9 activation in post-initiation phases of pulmonary oxygen toxicity. This concept is supported by recent findings that mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury [The reason why the activation of caspases did not induce apoptosis of TIIcells is not clear. On the one hand, activation of NF-κB has often been implicated as an anti-apoptotic event -45, and g injury . In factRecently, it has been shown that in response to severe hyperoxia of mice apoptosis and necrosis contribute to an extensive cell death; p53, bax, bcl-x, and Fas increased in mRNA and protein level, but the activity of caspase 3 and caspase 1 did not change . This inIn summary, TIIcells were not killed in the initiation phase of pulmonary oxygen toxicity. More precisely, its pro-apoptotic sensitisation is the background that, together with an additional stress factor, hyperoxia causes lung injury probably by apoptotic elimination of TIIcells. In agreement with this idea, we showed that the combination of the stress factors hyperoxia and vitamin E deficiency increases TIIcell apoptosis . Taking In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFα effect in vivo by anti-TNFα antibodies prevents the pro-apoptotic sensitisation of TIIcells.DAPI – 4',6-diamidino-2-phenylindole; EMSA – electrophoretic mobility shift assay; GSH – gluthatione; GSSG – oxidised gluthatione; NF-κB – nuclear factor-κB; TIIcell – type II pneumocyte; TNFα – tumour necrosis factor α; TNFR – TNFα receptor; TUNEL – terminal transferase dUTP nick end labellingHW carried out the immunohistochemistry, CS performed the mRNA analysis, AT carried out the protein measurements and participated in data analysis, MR applicated anti TNFα antibodies intratracheally, FS participated in the mRNA analysis and the design of the study, FG and BR conceived of the study, participated in its design and co-ordination, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.


In the absence in the major Australian administrative health record collections of a direct measure of the socioeconomic status of the individual about whom the event is recorded, analysis of the association between the health status, use of health services and socioeconomic status of the population relies an area-based measure of socioeconomic status.This paper explores the reliability of the area of address  as a proxy measure for socioeconomic disadvantage. The Western Australian Data Linkage System was used to show the extent to which hospital inpatient separation rates for residents of Perth vary by socioeconomic status of area of residence, when calculated at various levels of aggregation of area, from smallest (Census Collection District) to largest . Results are also provided of the reliability, over time, of the address as a measure of socioeconomic status.There is a strong association between the socioeconomic status of the usual address of hospital inpatients at the smallest level in Perth, and weaker associations when the data are aggregated to larger areas. The analysis also shows that a higher proportion of people from the most disadvantaged areas are admitted to hospital than from the most well-off areas (13% more), and that these areas have more separations overall (47% more), as a result of larger numbers of multiple admissions.Of people admitted to hospital more than once in a five year period, four out of five had not moved address by the time of their second episode. Of those who moved, the most movement was within, or between, areas of similar socioeconomic status, with people from the most well off areas being the least likely to have moved.Postcode level and SLA level data provide a reliable, although understated, indication of socioeconomic disadvantage of area. The majority of Perth residents admitted to hospital in Western Australia had the same address when admitted again within five years. Of those who moved address, the majority had moved within, or between, areas of similar socioeconomic status.Access to data about individuals from the Western Australian Data Linkage System shows that more people from disadvantaged areas are admitted to a hospital, and that they have more episodes of hospitalisation. Were data to be available across Australia on a similar basis, it would be possible to undertake research of greater policy-relevance than is currently possible with the existing separations-based national database. The majority of work in Australia describing the association between the health status, use of health services and socioeconomic status of the population uses an area-based measure of socioeconomic status. It is necessary to use a proxy measure (the socioeconomic status of the population in the area) because there is no direct measure in the major administrative health record collections of the socioeconomic status of the individual about whom the event is recorded.However, the application of an area-based measure requires a number of assumptions, including that people who move do so between, or within, geographic areas of similar socioeconomic status; and that the (often large) areas used in these analyses provide a reliable indication of the socioeconomic status and health service utilisation of the individuals in the area about whom the event is recorded. Area level socioeconomic status can also be considered as an independent predictor. For example, an individual with low socioeconomic status in an area of higher socioeconomic status is more likely to have better health outcomes than their counterpart in an area of lower socioeconomic status ,2. This In relation to this latter point, Hyndman et al  found thAnother limitation of the majority of Australian health-related datasets is that they record events , rather than individuals.The analysis in this report uses the Western Australian Data Linkage System to explore the reliability of area data as a proxy for socioeconomic disadvantage when analysed for the relatively large geographic units often used in health-related research: it also addresses the limitations of using data about events, rather than individuals. It does this by examining the extent to which hospital inpatient separation rates vary, both overall and by socioeconomic status of area of residence, when calculated at various levels of aggregation, from Census Collection District (CD) – the smallest area level for which a measure of socioeconomic status is available – to the larger units of postcode and Statistical Local Area (SLA). Methods applied include the calculation of correlation coefficients and examination of hospital separation rates by quintile of socioeconomic disadvantage of area, separately for events and individuals.The report also examines the reliability of the socioeconomic status of the address over time, by examining the extent of change in socioeconomic status of area of residence for individuals with repeat hospital episodes over a five year period.The analysis shows that aggregating data to larger area reduces the gap between the index scores for the most disadvantaged and least disadvantaged areas, with the greatest impact on the scores for the most disadvantaged areas. This results in an understatement of the extent of disadvantage in the most disadvantaged areas, as well as an understatement in inequality between the most well off and the poorest areas.Over the five years from 1994 to 1998, a total of 358 948 residents of Perth were admitted to a hospital in Western Australia on one or more occasion, an average of 71 750 individuals admitted per annum. Just over half (53.6%) the individuals admitted were females; 46.4% were males.The rate of individuals admitted was 16.4% higher for females (247.6 separations per 1000 population) than for males, (212.7 separations per 1000 population)  those admitted to hospital had one admission over this period, and more than one third 36.0%) had between two and four admissions, together comprising 90.6% of those admitted  of those admitted once, compared with 59.7% of those admitted more than once. For males, the proportions were 46.4% and 40.3%, respectively.There were 1 665 308 separations of Perth residents from Western Australian hospitals, an average of 2.53 separations per person admitted over the five years from 1994 to 1998. Over half (55.1%) of the separations were of females and 44.9% were of males.Figure The main differences in the profiles of male and female separations are evident at the youngest ages , from ages 20 to 44 years  and from 50 to 79 years . The ages at which the highest rates of admissions of individuals and of multiple admissions (the gap between the separations and admitted profiles) occur are clearly visible in the chart.Unlike the rates for individuals admitted Table , above, As noted, the majority of the analysis by socioeconomic status undertaken in the health sector in Australia is area based, and uses the postcode or SLA as the unit of analysis. This raises the question of the extent to which area based analyses at the postcode or SLA level provide a reliable indication of the socioeconomic status and health service utilisation of the individuals admitted. This report explores the reliability of postcode or SLA level data by examining the extent to which rates of individuals admitted and separations vary when calculated at various levels of aggregation (CD, postcode and SLA). Ideally, the comparison would be between the socioeconomic status of individuals and of areas; however, the smallest area level for which a measure of socioeconomic status is available is the CD.Variation in the minimum and maximum Index of Relative Socio-economic Disadvantage (IRSD) scores when calculated at the CD, postcode and SLA level is striking and clearly shows the value of the smaller unit in area based analyses Table . The ranThus, the use of larger area aggregates reduces the gap between the index scores for the most disadvantaged and least disadvantaged areas , with the greatest impact on the scores for the most disadvantaged areas . Notably, the difference between the maximum and minimum scores, and the absolute level of the scores, is much less marked between the postcode and SLA.There was a strong association between the IRSD scores for CDs and those for postcode of usual address at the first admission (a Spearman correlation coefficient of 0.74). The correlations were between CDs grouped to quintiles and postcodes grouped to quintiles, ranked by the IRSD, and not between individual CDs and postcodes. A weaker association was found between the quintiles for CDs and those for SLAs (0.64 for people with one separation and 0.63 for people with more than one separation)  than at the CD level   those admitted to hospital had one separation over this period, the proportion varied from 56.3% in Quintile 1 to 51.9% in Quintile 5 Table . This isThere is a substantial difference in the proportion of the population in Quintiles 5 and 1 having two or more separations (a difference of 38.8%, from a rate of 30 389 separations per 100 000 persons in Quintile 5 to 21 897 separations per 100 000 persons in Quintile 1): the differential for people having one separation is lower, although still notable at 16.1% (a rate of 32 790 separations per 100 000 persons in Quintile 5 and 28 231 separations per 100 000 persons in Quintile 1)  provide a reliable indication as to the socioeconomic status and use of health services of the individuals in the area.Data from the 1996 Census show that 53.5% of Perth's population at the 1996 Census reported that they had a different address to that at the previous Census, five years earlier [Similarly, almost four out of five people admitted to hospital more than once in a five year period had not moved (out of the CD of their address at the first separation) by the time of their second separation. For example, of the 298 809 people admitted to a Perth hospital more than once over the five year period 1994 to 1998, over three quarters (78.6%, 64 075 people) had the same address at the time of the second separation. People were recorded as having 'moved' if the CD of their address changed between the first and last separation over the period from 1994 to 1998. Movement to a different address within a CD was not included.The following table illustrates, for people with multiple admissions, the extent of movement by socioeconomic status. For this part of the analysis, the CD of first and last separation have been allocated to quintiles of socioeconomic disadvantage of area, to provide a comparison of the extent of movement between different levels of socioeconomic status. The construction of the quintiles is described in the section titled 'Methods, Measurement of socioeconomic status' under 'Methods.'Table • people from the most well off areas are less likely to have moved to areas of greatly different socioeconomic status (ie, changed quintiles) than are those from the most disadvantaged areas – 40.2% of people in the most advantaged areas (Quintile 1) remained there, despite moving from the CD of their first separation. The proportion in the most disadvantaged (Quintile 5) areas was a lower 30.5%;• while there is movement right across the socioeconomic profile, most movement is between adjacent quintiles. For example, of the 18 875 people who lived in the most disadvantaged areas at their first separation (and moved before a subsequent admission), 71.2% had moved to a CD in the same or next ranked quintile (Quintiles 5 or 4), with just 4.6% moving to the most advantaged areas. Similarly, of the 9 537 people in the most well off areas at their first separation, 63.0% had moved to a CD in the same or next ranked quintile (Quintiles 1 or 2), with a similarly low proportion (4.7%) moving to the most disadvantaged areas;• the most substantial movement between quintiles was of people moving from an address rated as Quintile 5 to one rated as Quintile 4 (40.7%); this was marginally higher than the proportions moving within Quintiles 4 or 1 (40.3% and 40.2%, respectively).There is a strong association between the quintile of socioeconomic disadvantage of area at the first and the last discharge when analysed by CD (a correlation coefficient of 0.88) or SLA (a correlation coefficient of 0.89) of usual address Table . This suThe analysis shows that, for Perth residents admitted to hospital, the use of larger area aggregates reduces the gap between the index scores for the most disadvantaged and least disadvantaged areas, thus understating the extent of inequality between these areas. The greatest impact of aggregation of areas is on the scores for the most disadvantaged areas. This results in an understatement of the extent of disadvantage in the most disadvantaged areas, as well as an understatement in the extent of inequality between the most well off and the poorest areas.Further, the analysis shows that a more people from the most disadvantaged areas are admitted to hospital than from the most well-off areas (13% more), and that these people have more separations overall (47% more), as a result of larger numbers of multiple admissions.As regards the extent of movement, four out of five people admitted to hospital more than once in a five year period had not moved (out of the CD of their address at the first separation) by the time of their second separation. In addition:• people from the most well off areas are less likely to have moved to areas of greatly different socioeconomic status than are those from the most disadvantaged areas;• while there is movement right across the socioeconomic profile, most movement out of a quintile is to areas in adjacent quintiles; and• the most substantial movement between quintiles was of people moving from an address rated as Quintile 5 to an address rated as Quintile 4, although this was only marginally higher than the proportions moving within Quintiles 4 or 1.In summary, postcode level and SLA level data provide a reliable indication of socioeconomic disadvantage of area, when compared with CD-level data. That is, the association between rates of total separations and individuals admitted and socioeconomic disadvantage of area evident at the smallest area level (CD) is also evident in the higher level area aggregates of postcode and SLA.It is reasonable to assume that similar relationships exist in other Australian cities, as well as in other health-related activity .Given the widespread use in Australia of area based analyses at the postcode and SLA level, and the limitations of CDs an area level for the analysis of most health datasets, it is important to know that such analyses provide a reliable indication of the direction and underlying strength of the influence of socioeconomic factors in hospital admissions rates.This is not to imply that the postcode or SLA are the ideal areal unit for analysis, nor that data for Collection District would be. The ideal population size for area-level analysis is likely to vary dependent on the number of cases in the dataset under analysis. For datasets with a large number of cases per capita  the number will be smaller than those with a small number of cases per capita (eg. deaths), even with aggregation of data over a number of years. May SLAs have much larger populations than are necessary to produce reliable results; and the populations of most CDs are too small  for individuals in the Western Australian Data Linkage System to the Australian Bureau of Statistics Population Census has the potential to add considerable value to such analyses. For example, it would be possible to examine an individual's characteristics of education, occupation, labour force status, housing tenure etc., and to more directly examine the relationships between the number of individuals admitted and total separations and these important socioeconomic variables. Linkage to death registration data would also be valuable in understanding more about outcomes related to socioeconomic status. This latter example is a possibility under recently announced plans for the ABS to test the linking of 2006 Census of Housing and Population data to other datasets, such as deaths registrations, held under their Act. This is similar to the approach elsewhere, including New Zealand . It is tThe report addresses differences in the number of individuals admitted and the number of separations they incurred. These are described as 'individuals', or individuals admitted' and separations . 'Separation' is the term describing a completed hospital episode: it is defined in the section titled 'Glossary, Separation' under 'Glossary.'Details of all separations to public and private hospitals in Western Australia for the five years from 1994 to 1998 were extracted from the Western Australian Hospital Morbidity Database (HMDS). Any separation records thought to belong to the same person had previously been linked together within the Data Linkage System, permitting analyses to be performed for both separations and individual persons. The population used in calculating rates is the 1996 Census population.The analysis has been limited to separations of residents of Perth, but includes separations occurring at any public acute or private hospitals in Western Australia.Areas used in the analysis are the Census Collection District (CD), postcode and Statistical Local Area (SLA). See Glossary for definitions of CD, postal area and SLA.The HMDS includes address details for each separation from a hospital in Western Australia since 1993. These addresses have been linked to a Western Australian street address database to assign northing and easting points (geo-codes). These points were then assigned to the appropriate 1991 or 1996 CD using the ABS CData96 mapping tool. The postcode and SLA of the address were then determined by allocation of CDs to postcode or SLA. The boundaries for CDs and SLAs are consistent. However, boundaries for CDs and postcodes are not, so CDs were allocated to postcodes on a 'best fit' basis (see Glossary).Consequently, comparisons can be made between results for CDs and postcode areas, CDs and SLAs and postcode areas and SLAs. This is particularly important, as much of the area analysis undertaken in the health sector in Australia uses the postcode or the SLA, as a majority of data are only available at these area levels, and it is widely accepted that the larger the area, the less homogenous the population is likely to be.There were 2 297 CDs in Perth at the 1996 Census, with 105 postcodes and 37 SLAs. The average population size at each of these area levels is shown in Table In the absence of any direct measure of socioeconomic status in the hospital inpatient data, the socioeconomic status of the area of the address of the individual admitted is used as a proxy measure. The Index of Relative Socio-Economic Disadvantage (IRSD) is the measure used to provide the socioeconomic status of the area of the address. The IRSD is one of five Socio-Economic Indexes for Areas (SEIFA) produced by the Australian Bureau of Statistics (ABS) from data collected at the 1996 Population Census. It is calculated at the CD level and can be produced for other area levels. The postcode and SLA level index scores in this report are the population weighted average of the IRSD scores for the CDs in the postcode or SLA. This calculation is undertaken for all CDs in the postcode or SLA, not just those for which hospital episodes were recorded.Each area level (CD, postcode or SLA) was allocated to one of five groups (quintiles). For example, for SLAs, Quintile 1 comprises the SLAs with the highest IRSD scores (most advantaged areas), and Quintile 5 comprises the SLAs with the lowest IRSD score (most disadvantaged areas): each quintile comprises approximately 20% of the Perth population. This process does not provide an exact allocation of population, so the resultant populations are only 'approximately' equal, and the larger the areal units being allocated, the less likely they are to be equal. As shown in Table Three (different) IRSD scores were added to each hospital separation record, based on the CD, postcode or SLA that had been previously assigned to the address on that record. It should be noted that these IRSD scores were actually the average score for the particular CD, postcode or SLA as calculated from 1996 Census data. Quintile ranks for each aggregation level were also applied using population weighting as described above.For analyses involving multiple admissions, the IRSD value used was that for the first separation in the five-year period. These 'first' separations were isolated using the internal links between separation records for the same person and the separation date. Of course, many of these 'first' separations could have been preceded by separations occurring before 1994.Rates are crude rates, per 100 000 population. Ideally the data would have been standardised (by the indirect method). However, access to the source data were limited and to requested tables, and standardisation was not an option.As the data were from a complete enumeration , confidence intervals were only calculated for measures of difference (in this case, rate ratios).The Spearman Rank Correlation has been used in the analysis to indicate the degree of correlation between pairs of variables.The Collection District (CD) is the smallest area level in the Australian Bureau of Statistics' statistical geography and is primarily an area used in the five yearly population census.The Index of Relative Socio-Economic Disadvantage (IRSD) is one of five Socio-Economic Indexes for Areas produced by the Australian Bureau of Statistics at recent population censuses. Produced using Principal Components Analysis, it summarises information available from variables related to education, occupation, income, family structure, race (the proportion of Indigenous people), ethnicity (poor proficiency in use of the English language) and housing. The variables are expressed as percentages of the relevant population. The IRSD is available at the Census Collection District level and was then be calculated for postcodes and SLAs by weighting the CD level scores by their population. The IRSD is calculated to show the relativity of areas to the Australian average for the particular set of variables which comprise it; this average score is set at 1000. Scores below 1000 indicate areas with relative disadvantaged populations under this measure, and scores above 1000 indicate areas with relatively advantaged populations. The IRSD scores at the Census Collection District (CD) level have been grouped to postal area, an area developed by ABS for the presentation of population counts and other Census data from the five-yearly population censuses to approximate postcode areas, as the ABS does not collect the postcode at the Census.The term describing a completed hospital episode is a 'separation'. At the time of admission to hospital, the age, sex, address of usual residence and other personal details of the patient are recorded. At the end of the episode, at the time of separation from hospital, details of the episode itself are recorded, including the date, time and method of separation . Consequently, hospital inpatient data collections are based on separations.The postal area is an area developed by ABS for the presentation of population counts and other Census data from the five-yearly population censuses. It approximates postcode areas, as the ABS does not collect the Australia Post postcode at the Census. Postal areas comprise Census Collection Districts (CDs) grouped to approximate postcode areas. Where a CD does not fit entirely within a postcode area, it is allocated to the postcode area into which the population largely falls. Where a CD covers more than one postcode area, the total CD population is allocated to one postcode.The IRSD scores at the Census Collection District (CD) level have been allocated to postal areas as described in the section titled 'Methods, Index of Relative Socio-Economic Disadvantage' under 'Methods.'. Similarly, the postal area of each separation was approximated from the CD of the address.The term postcode, rather than postal area, is used in the text, for ease of reading.See postal area, above.See section titled 'Methods, Measurement of socioeconomic status' under 'Methods.'An SLA in Perth is generally equivalent to a local government area, with additional codes allocated to local government areas split for statistical purposes .