Exploratory study of the 'weekend effect' for acute medical admissions to public hospitals in Queensland, Australia.

AIMS: To determine whether in-hospital deaths of patients admitted through emergency departments with acute exacerbations of chronic obstructive pulmonary disease (COPD), acute myocardial infarction, intracerebral haemorrhage and acute hip fracture are increased by weekend versus weekday admission (the 'weekend effect'). METHODS: We performed a retrospective analysis of statewide administrative data from public hospitals in Queensland, Australia, during the 2002/2003-2006/2007 financial years. The primary outcome was 30-day in-hospital mortality. The secondary outcome of 2-day in-hospital mortality helped determine whether increased mortality of weekend admissions was closely linked to weekend medical care. RESULTS: During the study period, there were 30 522 COPD, 17 910 acute myocardial infarction, 4183 acute hip fracture and 1781 intracerebral haemorrhage admissions. There was no significant weekend effect on 30-day in-hospital mortality for COPD (adjusted risk ratio = 0.92, 95% CI: 0.81-1.04, P= 0.222), intracerebral haemorrhage (adjusted risk ratio = 1.01, 95% CI: 0.86-1.16, P= 0.935) or acute hip fracture (adjusted risk ratio = 0.78, 95% CI: 0.54-1.03, P= 0.13). There was a significant weekend effect for acute myocardial infarction (adjusted risk ratio = 1.15, 95% CI: 1.03-1.26, P= 0.007). Two-day in-hospital mortality showed similar results. CONCLUSION: This is the first Australian study on the 'weekend effect' (in a cohort other than neonates), and the first study worldwide to assess specifically the weekend effect among COPD patients. Observed patterns were consistent with overseas research. There was a significant weekend effect for myocardial infarction. Further research is needed to determine whether location (e.g. rural), clinical (e.g. disease severity) and service provision factors (e.g. access to invasive procedures) influence the weekend effect for acute medical conditions in Australia.

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

The effects of exercise on skeletal muscle in the aged.

Age-related sarcopenia leads to muscle weakness and a concomitant increase in gait problems and the risk of hip fracture due to falling in the elderly. Muscle weakness reduces general activity levels in elderly individuals which in turn elevates the risk of osteoporosis due to a decrease in overall mechanical loading of the skeleton. At the same time, age-related sarcopenia is also linked to an increase in the risk of metabolic disorders such as adult onset (Type II) diabetes. However, it is widely accepted that increased mechanical loading of the musculoskeletal system (e.g., resistive exercise) can have a beneficial effect on both skeletal muscle and the supporting skeleton resulting in a significant reduction in the risk of developing all of the above age-related problems. As such, unloading models that exhibit many if not all of the same responses observed in aged muscle, including the capacity of exercise to reverse these responses, may provide valuable insight into the skeletal muscle aging process.

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor beta1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.

The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events.

Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma.

PURPOSE: To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC). METHODS: Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites. RESULTS: In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%). CONCLUSIONS: Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.

In situ localization of cholesterol in skeletal muscle by use of a monoclonal antibody.

A common perception is that cholesterol, the major structural lipid found in mammalian membranes, is localized nearly exclusively to the plasma membrane of living cells and that it is found in much smaller quantities in internal membranes. This perception is based almost exclusively on cell fractionation studies, in which density gradient centrifugation is used for purification of discrete subcellular membrane fractions. Here we describe a monoclonal antibody, MAb 2C5-6, previously reported to detect purified cholesterol in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM, Blanchette-Mackie EJ, and Alving CR. Proc Natl Acad Sci USA 85: 1902-1906, 1988), that is capable of detecting cholesterol in situ in the membranes of skeletal muscle sections. Localization of cholesterol, the dihydropyridine receptor of the T tubule, and the Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA2) by means of double and triple immunostaining protocols clearly demonstrates that cholesterol is primarily localized to the sarcoplasmic reticulum membranes of skeletal muscle rather than the sarcolemmal or T tubule membranes. The availability of this reagent and its ability to spatially localize cholesterol in situ may provide a greater understanding of the relationship between membrane cholesterol content and transmembrane signaling in skeletal muscle.

Perimembrane localization of the estrogen receptor alpha protein in neuronal processes of cultured hippocampal neurons.

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) alpha or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERalpha were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERalpha, rabbit polyclonal IgG antibody which does not recognize ERbeta was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERalpha, but abundant labeling for ERalpha throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 microM antisense oligonucleotide directed against the translation start site of ERalpha reduced ERalpha immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERalpha. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERalpha in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERalpha as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms.

Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

Plasma membrane ordering agent pluronic F-68 (PF-68) reduces neurotransmitter uptake and release and produces learning and memory deficits in rats.

A substantial body of evidence indicates that aged-related changes in the fluidity and lipid composition of the plasma membrane contribute to cellular dysfunction in humans and other mammalian species. In the CNS, reductions in neuronal plasma membrane order (PMO) (i.e., increased plasma membrane fluidity) have been attributed to age as well as the presence of the beta-amyloid peptide-25-35, known to play an important role in the neuropathology of Alzheimer's disease (AD). These PMO increases may influence neurotransmitter synthesis, receptor binding, and second messenger systems as well as signal transduction pathways. The effects of neuronal PMO on learning and memory processes have not been adequately investigated, however. Based on the hypothesis that an increase in PMO may alter a number of aspects of synaptic transmission, we investigated several neurochemical and behavioral effects of the membrane ordering agent, PF-68. In cell culture, PF-68 (nmoles/mg SDS extractable protein) reduced [3H]norepinephrine (NE) uptake into differentiated PC-12 cells as well as reduced nicotine stimulated [3H]NE release. The compound (800-2400 microg/kg, i.p., resulting in nmoles/mg SDS extractable protein in the brain) decreased step-through latencies and increased the frequencies of crossing into the unsafe side of the chamber in inhibitory avoidance training. In the Morris water maze, PF-68 increased the latencies and swim distances required to locate a hidden platform and reduced the time spent and distance swam in the previous target quadrant during transfer (probe) trials. PF-68 did not impair performance of a well-learned working memory task, the rat delayed stimulus discrimination task (DSDT), however. Studies with 14C-labeled PF-68 indicated that significant (pmoles/mg wet tissue) levels of the compound entered the brain from peripheral (i.p.) injection. No PF-68 related changes were observed in swim speeds or in visual acuity tests in water maze experiments, rotorod performance, or in tests of general locomotor activity. Furthermore, latencies to select a lever in the DSDT were not affected. These results suggest that PF-68 induced deficits in learning and memory without confounding peripheral motor, sensory, or motivational effects at the tested doses. Furthermore, none of the doses induced a conditioned taste aversion to a novel 0.1% saccharin solution indicating a lack of nausea or gastrointestinal malaise induced by the compound. The data indicate that increases in neuronal plasma membrane order may have significant effects on neurotransmitter function as well as learning and memory processes. Furthermore, compounds such as PF-68 may also offer novel tools for studying the role of neuronal PMO in mnemonic processes and changes in PMO resulting from age-related disorders such as AD.

Bed rest decreases mechanically induced myofiber wounding and consequent wound-mediated FGF release.

Using a terrestrial model of spaceflight (i.e., bed rest), we investigated the amount of myofiber wounding and fibroblast growth factor (FGF) release that occurs during unloading. Myofiber wounding was determined by serum levels of the creatine kinase MM (CKMM) isoform before and after bed rest. Serum levels of both acidic FGF (aFGF) and basic FGF were also determined. A second group of subjects was treated in an identical fashion except that they underwent a resistive exercise program during bed rest. Bed rest alone caused significant (P < 0.05; n = 7) reductions in post-bed-rest serum levels of both CKMM and aFGF, which were paralleled by a significant (P < 0.05; n = 7) decrease in myofiber size. In contrast, bed rest plus resistive exercise resulted in significant (P < 0.05; n = 7) increases in post-bed-rest serum levels of both CKMM and aFGF, which were paralleled by inhibition of the atrophic response. These results suggest that mechanically induced, myofiber wound-mediated FGF release may play an important role in the etiology of unloading-induced skeletal muscle atrophy.

Impact of resistance exercise during bed rest on skeletal muscle sarcopenia and myosin isoform distribution.

Because resistance exercise (REx) and bed-rest unloading (BRU) are associated with opposing adaptations, our purpose was to test the efficacy of REx against the effects of 14 days of BRU on the knee-extensor muscle group. Sixteen healthy men were randomly assigned to no exercise (NoEx; n = 8) or REx (n = 8). REx performed five sets of leg press exercise with 80-85% of one repetition maximum (1 RM) every other day during BRU. Muscle samples were removed from the vastus lateralis muscle by percutaneous needle biopsy. Myofiber distribution was determined immunohistochemically with three monoclonal antibodies against myosin heavy chain (MHC) isoforms (I, IIa, IIx). MHC distribution was further assessed by quantitative gel electrophoresis. Dynamic 1-RM leg press and unilateral maximum voluntary isometric contraction (MVC) were determined. Maximal neural activation (root mean squared electromyogram) and rate of torque development (RTD) were measured during MVC. Reductions (P < 0.05) in type I (15%) and type II (17%) myofiber cross-sectional areas were found in NoEx but not in REx. Electrophoresis revealed no changes in MHC isoform distribution. The percentage of type IIx myofibers decreased (P < 0.05) in REx from 9 to 2% and did not change in NoEx. 1 RM was reduced (P < 0.05) by 9% in NoEx but was unchanged in REx. MVC fell by 15 and 13% in NoEx and REx, respectively. The agonist-to-antagonist root mean squared electromyogram ratio decreased (P < 0.05) 19% in REx. RTD slowed (P < 0.05) by 54% in NoEx only. Results indicate that REx prevented BRU-induced myofiber atrophy and also maintained training-specific strength. Unlike spaceflight, BRU did not induce shifts in myosin phenotype. The reported benefits of REx may prove useful in prescribing exercise for astronauts in microgravity.

Mechanical load induces sarcoplasmic wounding and FGF release in differentiated human skeletal muscle cultures.

The transduction mechanism (or mechanisms) responsible for converting a mechanical load into a skeletal muscle growth response are unclear. In this study we have used a mechanically active tissue culture model of differentiated human skeletal muscle cells to investigate the relationship between mechanical load, sarcolemma wounding, fibroblast growth factor release, and skeletal muscle cell growth. Using the Flexcell Strain Unit we demonstrate that as mechanical load increases, so too does the amount of sarcolemma wounding. A similar relationship was also observed between the level of mechanical load inflicted on the cells and the amount of bFGF (FGF2) released into the surrounding medium. In addition, we demonstrate that the muscle cell growth response induced by chronic mechanical loading in culture can be inhibited by the presence of an antibody capable of neutralizing the biological activity of FGF. This study provides direct evidence that mechanically induced, sarcolemma wound-mediated FGF release is an important autocrine mechanism for transducing the stimulus of mechanical load into a skeletal muscle growth response.

Contraction-induced cell wounding and release of fibroblast growth factor in heart.

The heart hypertrophies in response to certain forms of increased mechanical load, but it is not understood how, at the molecular level, the mechanical stimulus of increased load is transduced into a cell growth response. One possibility is that mechanical stress provokes the release of myocyte-derived autocrine growth factors. Two such candidate growth factors, acidic and basic fibroblast growth factor (aFGF and bFGF, respectively), are released via mechanically induced disruptions of the cell plasma membrane. In the present study, we demonstrate that transient, survivable disruption (wounding) of the cardiac myocyte plasma membrane is a constitutive event in vivo. Frozen sections of normal rat heart were immunostained to reveal the distribution of the wound event marker, serum albumin. Quantitative image analysis of these sections indicated that an average of 25% of the myocytes contained cytosolic serum albumin; ie, this proportion had suffered a plasma membrane wound. Wounding frequency increased approximately threefold after beta-adrenergic stimulation of heart rate and force of contraction. Heparin-Sepharose chromatography, enzyme-linked immunosorbent assay, growth assay coupled with antibody neutralization, and two-dimensional SDS-PAGE followed by immunoblotting were used to demonstrate that both aFGF and bFGF were released from an ex vivo beating rat heart. Importantly, beta-adrenergic stimulation of heart rate and force of contraction increased FGF release. Cell wounding is a fundamental but previously unrecognized aspect of the biology of the cardiac myocyte. We propose that contraction-induced cardiac myocyte wounding releases aFGF and bFGF, which then may act as autocrine growth-promoting stimuli.

Cytoplasmic loading of dyes, protein and plasmid DNA using an impact-mediated procedure.

We describe a method and apparatus designed to rapidly and reproducibly produce transient, survivable plasma membrane disruptions--"wounds"--in order to gain access to the cytoplasm of eukaryotic cells growing in culture. Compressed gas is used to propel glass beads, dispersed as a uniform aerosol, at adherent cells growing on a culture substratum. The impact of beads with the cells creates plasma membrane wounds. Macromolecules, such as dyes, proteins and plasmid DNAs, diffuse from the extracellular environment directly into the cytoplasmic compartment of the cell through these wounds. Resealing of the plasma membrane, necessary for cell survival, traps macromolecules within the cytoplasm of the cell.

Nicotine stimulation of nerve growth factor receptor expression.

Previous studies have suggested that nicotine may have beneficial actions in neurodegenerative disease models. The purpose of the experiments described in this study was to determine whether the long lasting and beneficial effects of nicotine observed previously could be expressed through actions upon nerve growth factor (NGF) receptors. Using a differentiated PC-12 neuronal cell model, we have detected an increase in expression of cell surface NGF receptor protein after acute exposure to nicotine in the micromolar range. In addition, we have also observed a persistent effect upon NGF receptor expression which lasted even after nicotine (nanomolar range) was removed from the tissue culture medium. This increase in cell surface NGF receptor protein was blocked in the presence of mecamylamine, indicating that this effect is likely nicotinic receptor mediated. These results are consistent with the hypothesis that the lasting and beneficial actions of nicotine previously observed in vivo may involve an indirect effect upon the level of neuronal cell surface NGF receptor expression. Our observations offer one possible mechanism for a potential neurotrophic effect of nicotine.

Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle.

Using muscle as an in vivo model system, we have tested the hypothesis that basic fibroblast growth factor is released from a cytoplasmic storage site into the extra-cellular environment via diffusion through survivable, mechanically-induced plasma membrane disruptions. Normal and dystrophic (mdx) mouse muscle were studied. Strong immunostaining for bFGF was detected in the cytoplasm of myofibers of uninjured muscle fixed in situ by perfusion. By contrast, myofibers did not stain cytoplasmically for bFGF after suffering lethal disruptions of their plasma membranes caused by freezing and thawing followed by sectioning. Sub-lethal, transient disruptions of myofiber plasma membranes--termed plasma membrane 'wounds'--were shown to be induced by needle puncture or exercise of muscle. Quantitative image analysis revealed that these wounded fibers contained significantly reduced levels of bFGF. Dystrophic exercised and unexercised muscle was found to possess an approximately 6-fold higher proportion of wounded myofibers than does normal muscle under equivalent conditions. Release of bFGF at a rate that is a direct function of the frequency of myofiber wounding may explain in part how a muscle adjusts its growth to meet changing mechanical demand as well as the pathological hypertrophy characteristic of certain stages of muscular dystrophy.

Syringe loading introduces macromolecules into living mammalian cell cytosol.

We describe a simple, efficient, gentle and inexpensive technique for the introduction of normally impermeant macromolecules into the cytosol of living mammalian cells growing in suspension or attached to the culturing substratum. Loading is achieved by the production of transient, survivable plasma membrane disruptions as cells are passed back and forth through a standard syringe needle or similar narrow orifice. The loading volume required, which contains cells and the macromolecule to be loaded, can be as little as 5 microliters, thus minimizing the use of valuable reagents. In addition, we report that the surfactant molecule, Pluronic F-68, is capable of altering the physical properties of the plasma membrane in such a way as to increase loading efficiency and the long-term survivability of cells loaded by this and other mechanically based cell-loading techniques.