Markers of inflammation: data from the MOSAIC randomised trial of CPAP for minimally symptomatic OSA.

UNLABELLED: The Multi-centre Obstructive Sleep Apnoea Interventional Cardiovascular (MOSAIC) trial compared 6 months of CPAP therapy, versus no CPAP, in 391 patients with minimally symptomatic obstructive sleep apnoea (OSA). We now report some exploratory outcomes, markers of systemic inflammation (interleukin 6 (IL-6), IL-10, C reactive protein, tumour necrosis factor). We found no consistent changes (all p values >0.13). TRIAL REGISTRATION NUMBER: ISRCTN 34164388.

Cardiovascular event rates in the MOSAIC trial: 2-year follow-up data.

The Multicentre Obstructive Sleep Apnoea Intervention Cardiovascular (MOSAIC) trial investigated the effect of continuous positive airway pressure (CPAP) on both sleepiness and predicted cardiovascular risk over 6 months in minimally symptomatic patients with obstructive sleep apnoea. Although there was clear benefit in terms of Epworth Sleepiness Score, there was no improvement in blood pressure and predicted vascular risk score. In order to calculate the required size of future trials, with real vascular events as the endpoint, the rate of such events in this population is needed. 188 patients from the original trial were followed for 2 years. The overall number of new vascular events over the 2 years was 25, and all-cause mortality was 4. There was a weak statistically significant reduction in vascular events in the CPAP group (p=0.049). Large-scale randomised trials are needed to determine if CPAP causes a real reduction in vascular events in minimally symptomatic patients. Based on our figures, future trials of CPAP versus no treatment would need to randomise approximately 2540 patients to not miss a real reduction in vascular events and over 6000 for mortality.

Internal rotational error of the tibial component is a major cause of pain after total knee replacement.

This study used CT analysis to determine the rotational alignment of 39 painful and 26 painless fixed-bearing total knee replacements (TKRs) from a cohort of 740 NexGen Legacy posterior-stabilised and cruciate-retaining prostheses implanted between May 1996 and August 2003. The mean rotation of the tibial component was 4.3 degrees of internal rotation (25.4 degrees internal to 13.9 degrees external rotation) in the painful group and 2.2 degrees of external rotation (8.5 degrees internal to 18.2 degrees external rotation) in the painfree group (p = 0.024). In the painful group 17 tibial components were internally rotated more than 9 degrees compared with none in the painfree group (p < 0.001). Additionally, six femoral components in the painful group were internally rotated more than 6 degrees compared with none in the painfree group (p = 0.017). External rotational errors were not found to be associated with pain. Overall, 22 (56.4%) of the painful TKRs had internal rotational errors involving the femoral, the tibial or both components. It is estimated that at least 4.6% of all our TKRs have been implanted with significant internal rotational errors.

The prevalence of retinopathy in men with Type 2 diabetes and obstructive sleep apnoea.

AIMS: To clarify the relationship between obstructive sleep apnoea (OSA) and diabetic retinopathy. RESEARCH DESIGN AND METHODS: A cohort of 240 men from primary and secondary care previously participated in a study on the prevalence of OSA in Type 2 diabetes and provided anthropometric information, details of their diabetes, had glycated haemoglobin (HbA1c) measured and overnight oximetry performed. They were re-contacted for permission to review their routine screening clinical retinal photographs, which were then scored by a trained grader, providing detailed retinopathy, maculopathy and photocoagulation scores. RESULTS: One hundred and eighteen men both consented and had retinal photographs available to review. Of these, 24% had OSA, with mean+/-sd 4% oxygen saturation (SaO2) dips/h of 20.9+/-16.6 vs. 2.8+/-2.1 in the non-OSA group. As expected, the OSA group had a significantly higher mean body mass index of 31.9+/-5.2 vs. 28.5+/-5.1 kg/m2 and neck size 44.5+/-3.6 vs. 41.9+/-2.5 cm, but the two groups did not differ significantly in age, diabetes duration, diabetes treatment, HbA1c, smoking history or proportion with known hypertension. Retinopathy and maculopathy scores were significantly worse in the OSA group (P<0.0001). Multiple regression analysis showed only OSA (R2=0.19, P<0.0001) and HbA1c (R2=0.04, P=0.03) to be significant independent predictors of retinopathy. OSA was the only independent significant predictor of the total microaneurysm score (R2=0.21, P=0.004), a detailed retinopathy subclassification. OSA was the only independent significant predictor of maculopathy (R2=0.3, P<0.001). CONCLUSION: In men with Type 2 diabetes, there is a strong association between retinopathy and OSA, independent of conventional retinopathy risk factors.

Circulating cell-derived microparticles in patients with minimally symptomatic obstructive sleep apnoea.

Moderate-severe obstructive sleep apnoea (OSA) has been associated with several pro-atherogenic mechanisms and increased cardiovascular risk, but it is not known if minimally symptomatic OSA has similar effects. Circulating cell-derived microparticles have been shown to have pro-inflammatory, pro-coagulant and endothelial function-impairing effects, as well as to predict subclinical atherosclerosis and cardiovascular risk. In 57 patients with minimally symptomatic OSA, and 15 closely matched control subjects without OSA, AnnexinV-positive, platelet-, leukocyte- and endothelial cell-derived microparticles were measured by flow cytometry. In patients with OSA, median (interquartile range) levels of AnnexinV-positive microparticles were significantly elevated compared with control subjects: 2,586 (1,566-3,964) microL(-1) versus 1,206 (474-2,501) microL(-1), respectively. Levels of platelet-derived and leukocyte-derived microparticles were also significantly higher in patients with OSA (2,267 (1,102-3,592) microL(-1) and 20 (14-31) microL(-1), respectively) compared with control subjects (925 (328-2,068) microL(-1) and 15 (5-23) microL(-1), respectively). Endothelial cell-derived microparticle levels were similar in patients with OSA compared with control subjects (13 (8-25) microL(-1) versus 11 (6-17) microL(-1)). In patients with minimally symptomatic obstructive sleep apnoea, levels of AnnexinV-positive, platelet- and leukocyte-derived microparticles are elevated when compared with closely matched control subjects without obstructive sleep apnoea. These findings suggest that these patients may be at increased cardiovascular risk, despite being minimally symptomatic.

Prevalence of obstructive sleep apnoea in men with type 2 diabetes.

BACKGROUND: A study was undertaken to establish the prevalence of obstructive sleep apnoea (OSA) in men with type 2 diabetes. METHODS: Men with type 2 diabetes from local hospital and selected primary care practitioner databases received questionnaires about snoring, apnoeas, and daytime sleepiness based on the Berlin questionnaire. Selected respondents had overnight oximetry to establish whether they had OSA. Comparisons of oximetry were made with those from a previous general population study. HbA1c results were collected. RESULTS: 1682 men were sent questionnaires, 56% of whom replied. 57% scored as "high" and 39% as "low" risk for OSA; 4% were already known to have OSA. Oximetry was performed in 240 respondents from both risk groups: 31% of the "high" and 13% of the "low" risk group had significant OSA (more than 10 >4% Sao(2) dips/hour or Sao(2) tracing consistent with OSA). These results were verified by detailed sleep studies. Extrapolation of the oximetry data to the questionnaire respondent population suggests that 23% had OSA. Comparison of the oximetry results with men from a previous general population study (using only more than 10 >4% Sao(2) dips/hour to define OSA) showed the prevalence of OSA is significantly higher in this diabetes population (17% v 6%, p<0.001). Multiple linear regression revealed BMI and diabetes as significant independent predictors of OSA. Following correction for BMI (which explained 13% of the variance in OSA), diabetes explained a further 8% of the variance (p<0.001). There was a low correlation between OSA severity and HbA1c in the subgroup recruited from the hospital database (r = 0.2, p = 0.006) which remained significant after allowing for obesity (p = 0.03). CONCLUSIONS: OSA is highly prevalent in men with type 2 diabetes; most are undiagnosed. Diabetes itself may be a significant independent contributor to the risk of OSA.

Immunohistochemical detection of the sodium-calcium exchanger in rat hippocampus cultures using subtype-specific antibodies.

All of the known Na+/Ca2+ exchanger subtypes, NCX1-3, are expressed in the brain, albeit with marked regional differences. On the mRNA level, overall expression seems most prominent for NCX2, intermediate for NCX1, and, except for a few regions, low for NCX3. Using three subtype-specific antibodies, we have now studied the cellular expression of the NCX subtypes in rat hippocampus cultures by immunohistochemical techniques. Our results provide evidence for a highly cell-specific expression pattern of NCX subtypes and show surprisingly little colocalization. NCX1 and NCX3 are both primarily expressed in neuronal cells. While NCX1 is found in the large majority of neurons, NCX3 expression was restricted to a small minority of cells. By contrast, NCX2 was almost exclusively present in glial cells. The NCX2 antibody, a IgM, stained glial cell membranes as well as an intermediate fibrillar system. In spite of extensive screening, the nature of this fiber system has not yet been identified.

An outbreak of methicillin resistant Staphylococcus aureus on a burn unit: potential role of contaminated hydrotherapy equipment.

OBJECTIVE: To report a multi-institution outbreak caused by a single strain of methicillin-resistant Staphylococcus aureus (MRSA). OUTBREAK: Between September 19 and November 20, 1996 an index case and five secondary cases of nosocomial MRSA occurred on a 26 bed adult plastic surgery/burn unit (PSBU) at a tertiary care teaching hospital. Between November 11 and December 23, 1996, six additional cases were identified at a community hospital. One of the community hospital cases was transferred from the PSBU. All strains were identical by pulsed-field gel electrophoresis. MRSA may have contributed to skin graft breakdown in one case, and delayed wound healing in others. Patients required 2 to 226 isolation days. CONTROL MEASURES: A hand held shower and stretcher for showering in the hydrotherapy room of the PSBU were culture positive for the outbreak strain, and the presumed means of transmission. Replacement of stretcher showering with bedside sterile burn wound compresses terminated the outbreak. The PSBU was closed to new admissions and transfers out for 11 days during the investigation. Seven of 12 patients had effective decolonization therapy. CONCLUSION: Environmental contamination is a potential source of nosocomial MRSA transmission on a burn unit. Notification among institutions and community care providers of shared patients infected or colonized with an antimicrobial resistant microorganism is necessary.

Excess of twins among affected sibling pairs with autism: implications for the etiology of autism.

It is widely accepted that genes play a role in the etiology of autism. Evidence for this derives, in part, from twin data. However, despite converging evidence from gene-mapping studies, aspects of the genetic contribution remain obscure. In a sample of families selected because each had exactly two affected sibs, we observed a remarkably high proportion of affected twin pairs, both MZ and DZ. Of 166 affected sib pairs, 30 (12 MZ, 17 DZ, and 1 of unknown zygosity) were twin pairs. Deviation from expected values was statistically significant (P<10(-6) for all twins); in a similarly ascertained sample of individuals with type I diabetes, there was no deviation from expected values. We demonstrate that to ascribe the excess of twins with autism solely to ascertainment bias would require very large ascertainment factors; for example, affected twin pairs would need to be, on average, approximately 10 times more likely to be ascertained than affected non-twin sib pairs (or 7 times more likely if "stoppage" plays a role). Either risk factors (related to twinning or to fetal development) or other factors (genetic or nongenetic) in the parents may contribute to autism.

A disulfide bond is required for functional assembly of NCX1 from complementary fragments.

The cardiac Na(+)-Ca(2+) exchanger consists of a single polypeptide with two transmembrane segment (TMS) clusters separated by a large intracellular loop between TMS5 and TMS6 (Nicoll et al. (1999) J. Biol. Chem. 274, 910-917; Iwamoto et al. (1999) FEBS Lett. 446, 264-268). A "split" exchanger can be expressed by dividing the exchanger cDNA into two fragments so that the NH(2)- and CO(2)H-terminal portions of the protein are expressed as separate polypeptides in HEK293 cells. Expression of partial exchanger molecules did not result in detectable exchanger activity. Cells coexpressing both portions of the exchanger, however, displayed between 30 and 50% of the activity of the intact wild-type exchanger. The full-length exchanger contains a disulfide bond between residues 14 or 20 and 792. We examined the role of this disulfide bond in the split exchanger by mutagenesis and expression studies. Our results indicate that the function of the exchanger requires both TMS clusters and that the C(14 or 20)/C792 disulfide bond is essential for expression of active exchangers from half molecules.

Methicillin-resistant Staphylococcus aureus: profiles oceans apart--Canadian and Saudi Arabian experiences.

Methicillin-resistant Staphylococcus aureus (MRSA), a pathogen which has increased over the past three decades, is responsible for nosocomial infections and adverse patient outcomes. It is a pathogen of global importance. Rates of patient colonization or infection vary greatly internationally. Lower rates have been observed in Canada and Saudi Arabia compared to the United States and United Kingdom. Although these lower rates may appear reassuring, the trend in MRSA observed in two capital cities, Winnipeg, Canada and Riyadh, Saudi Arabia are consistent with a widespread global increase in MRSA.

Helix packing of functionally important regions of the cardiac Na(+)-Ca(2+) exchanger.

In a revised topological model of the cardiac Na(+)-Ca(2+) exchanger, there are nine transmembrane segments (TMSs) and two possible re-entrant loops (Nicoll, D. A., Ottolia, M., Lu, Y., Lu, L., and Philipson, K. D. (1999) J. Biol. Chem. 274, 910-917; Iwamoto, T., Nakamura, T. Y., Pan, Y., Uehara, A., Imanaga, I., and Shigekawa, M. (1999) FEBS Lett. 446, 264-268). The TMSs form two clusters separated by a large intracellular loop between TMS5 and TMS6. We have combined cysteine mutagenesis and oxidative cross-linking to study proximity relationships of TMSs in the exchanger. Pairs of cysteines were reintroduced into a cysteine-less exchanger, one in a TMS in the NH(2)-terminal cluster (TMSs 1-5) and the other in a TMS in the COOH-terminal cluster (TMSs 6-9). The mutant exchanger proteins were expressed in HEK293 cells, and disulfide bond formation between introduced cysteines was analyzed by gel mobility shifts. Western blots showed that S117C/V804C, A122C/Y892C, A151C/T815C, and A151C/A821C mutant proteins migrated at 120 kDa under reducing conditions and displayed a partial mobility shift to 160 kDa under nonreducing conditions. This shift indicates the formation of a disulfide bond between these paired cysteine residues. Copper phenanthroline and the cross-linker N', N'-o-phenylenedimaleimide enhanced the mobility shift to 160 kDa. Our data suggest that TMS7 is close to TMS3 near the intracellular side of the membrane and is in the vicinity of TMS2 near the extracellular surface. Also, TMS2 must adjoin TMS8. This initial packing model of the exchanger brings two functionally important domains in the exchanger, the alpha 1 and alpha 2 repeats, close to each other.

Effects of growth hormone treatment on red cell plasma membrane fatty acid constituents in hypopituitary adults.

The effects of replacement with recombinant human GH (hGH) on red cell plasma membrane fatty acid and cholesterol constituents were assessed in nine adult patients with growth hormone deficiency. They were treated with hGH in a dose of 0.125 U.kg-1.wk-1 for four weeks and at 0.25 U.kg-1.wk-1 thereafter for an overall mean duration 13.5 +/- 3.9 months (mean +/- SD). The relative proportions of the various phospholipid fatty acid constituents and the proportion of cholesterol in the phospholipid bilayer were assayed every six months. At the end of the study, the percentage of arachadonic acid (20:4) in membrane phospholipid was found to rise by an average of 3.7% (P < 0.05) and there appeared to be a nonsignificant trend showing an increase in highly unsaturated fatty acids, namely linoleate (18:2) and gamma linolenic acid (18:3) and a corresponding decrease in unsaturated fatty acids, namely palmitate (16:0) and stearate (18:0) and monounsaturated fatty acids such as palmitoleic acid (16:1), oleic acid (18:1) and oleic acid isomer (18:1 iso). In addition, the proportion of cholesterol in the plasma membrane i.e. the cholesterol/phospholipid ratio was found to decrease by 0.84% (P < 0.05). There was a significant increase in HbAlc from 4.85 +/- 0.51 to 4.94 +/- 0.45% (P < 0.01) by the end of the study.

Sodium-calcium exchange: a molecular perspective.

Plasma membrane Na(+)-Ca2+ exchange is an essential component of Ca2+ signaling pathways in several tissues. Activity is especially high in the heart where the exchanger is an important regulator of contractility. An expanding exchanger superfamily includes three mammalian Na(+)-Ca2+ exchanger genes and a number of alternative splicing products. New information indicates that the exchanger protein has nine transmembrane segments. The exchanger, which transports Na+ and Ca2+, is also regulated by these substrates. Some molecular information is available on regulation by Na+ and Ca2+ and by PIP2 and phosphorylation. Altered expression of the exchanger in pathophysiological states may contribute to various cardiac phenotypes. Use of transgenic approaches is beginning to improve our knowledge of exchanger function.

Functional analysis of a disulfide bond in the cardiac Na(+)-Ca(2+) exchanger.

The electrophoretic mobility of the cardiac Na(+)-Ca(2+) exchange protein is different under reducing and nonreducing conditions. This mobility shift is eliminated in a cysteine-less exchanger, suggesting that the presence or absence of an intramolecular disulfide bond alters the conformation and mobility of the exchanger. Using cysteine mutagenesis and biochemical analysis, we have identified the cysteine residues involved in the disulfide bond. Cysteine 792 in loop h of the exchanger forms a disulfide bond with either cysteine 14 or 20 near the NH(2) terminus. Because the NH(2) terminus is extracellular, the data establish that loop h must also be extracellular. A rearrangement of disulfide bonds has previously been implicated in the stimulation of exchange activity by combinations of reducing and oxidizing agents. We have investigated the role of cysteines in the stimulation of the exchanger by the combination of FeSO(4) and dithiothreitol (Fe-DTT). Using the giant excised patch technique, we find that stimulation of the wild type exchanger by Fe-DTT is primarily due to the removal of a Na(+)-dependent inactivation process. Analysis of mutated exchangers, however, indicates that cysteines are not responsible for stimulation of the exchange activity by Fe-DTT. Ca(2+) blocks modification of the exchanger by Fe-DTT. Disulfide bonds are not involved in redox stimulation of the exchanger, and the modification reaction is unknown. Modulation of Na(+)-dependent inactivation may be a general mechanism for regulation of Na(+)-Ca(2+) exchange activity and may have physiological significance.

Cloning, expression, and characterization of the trout cardiac Na(+)/Ca(2+) exchanger.

Isoform 1 of the cardiac Na(+)/Ca(2+) exchanger (NCX1) is an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7 degrees C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout (Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca(2+) binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at approximately 23 degrees C demonstrated Na(+)-dependent inactivation and Ca(2+)-dependent activation in a manner qualitatively similar to that for NCX1 currents.

A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger.

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+ gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.

Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster.

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current-voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being approximately 80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced ( approximately 20-40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger.

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.