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Hereditary sensory neuropathy type 1 (HSAN I) is an autosomal dominant inherited neurodegenerative disorder of the peripheral nervous system associated with mutations in the SPTLC1 subunit of the serine palmitoyltransferase (SPT). Four missense mutations (C133W, C133Y, V144D and G387A) in SPTLC1 were reported to cause HSAN I. SPT catalyses the condensation of Serine and Palmitoyl-CoA, which is the first and rate-limiting step in the de novo synthesis of ceramides. Earlier studies showed that C133W and C133Y mutants have a reduced activity, whereas the impact of the V144D and G387A mutations on the human enzyme was not tested yet. In this paper, we show that none of the HSAN I mutations interferes with SPT complex formation. We demonstrate that also V144D has a reduced SPT activity, however to a lower extent than C133W and C133Y. In contrast, the G387A mutation showed no influence on SPT activity. Furthermore, the growth phenotype of LY-B cellsa SPTLC1 deficient CHO cell linecould be reversed by expressing either the wild-type SPTLC1 or the G387A mutant, but not the C133W mutant. This indicates that the G387A mutation is most likely not directly associated with HSAN I. These findings were genetically confirmed by the identification of a nuclear HSAN family which showed segregation of the G387A variant as a non-synonymous SNP.

Serine palmitoyltransferase (SPT) catalyzes the condensation of l-serine and palmitoyl-CoA, which is the rate-limiting step in the de novo synthesis of sphingolipids. SPT activity is commonly measured by monitoring the incorporation of radiolabeled l-serine into 3-ketodihydrosphingosine. In this article, we introduce several adaptations of the established protocol to improve sensitivity, reproducibility, and practicability of the assay. A significant improvement of this new protocol is the possibility to measure SPT activity in total cell lysate instead of microsomes. The assay is furthermore extended by the introduction of a nonradioactive, HPLC-based detection protocol. The suggested HPLC method offers several advantages, most importantly, a 20-fold lower detection limit compared with the radioactive assay and the possibility to use an internal standard to correct for variation in the extraction.

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation, and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human β-cells, LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of β-cells from wild-type and LDL receptor-deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-1β and glucose-induced apoptosis. IL-1β-induced β-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A1 (the main protein component of HDL), or sphingosine-1-phosphate (a bioactive sphingolipid mostly carried by HDL). In murine β-cells, the protective effect of HDL against IL-1β-induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type 1. Our data show that both LDL and HDL affect function or survival of β-cells and raise the question whether dyslipidemia contributes to β-cell failure and hence the manifestation and progression of type 2 diabetes mellitus.

RATIONALE: Reductions in mortality following improvements in air quality were documented by several studies, and our group found, in an earlier analysis, that decreasing particulate levels attenuate lung function decline in adults. OBJECTIVES: We investigated whether decreases in particulates with an aerodynamic diameter of less than 10 microm (PM10) were associated with lower rates of reporting respiratory symptoms (i.e., decreased morbidity) on follow-up. METHODS: The present analysis includes 7,019 subjects who underwent detailed baseline examinations in 1991 and a follow-up interview in 2002. Each subject was assigned model-based estimates of average PM10 during the 12 months preceding each health assessment and the difference was used as the exposure variable of interest (DeltaPM10). Analyses were stratified by symptom status at baseline and associations between DeltaPM10 and change in symptom status during follow-up were adjusted for important baseline characteristics, smoking status at follow-up, and season. We then estimated adjusted odds ratios for symptoms at follow-up and numbers of symptomatic cases prevented due to the observed reductions in PM10. MEASUREMENTS AND MAIN RESULTS: Residential exposure to PM10 was lower in 2002 than in 1991 (mean decline 6.2 microg/m3; SD = 3.9 microg/m3). Estimated benefits (per 10,000 persons) attributable to the observed changes in PM10-levels were: 259 (95% confidence interval [CI]: 102-416) fewer subjects with regular cough, 179 (95% CI, 30-328) fewer subjects with chronic cough or phlegm and 137 (95% CI, 9-266) fewer subjects with wheezing and breathlessness. CONCLUSIONS: Reductions in particle levels in Switzerland over the 11-year follow-up period had a beneficial effect on respiratory symptoms among adults.

A-type natriuretic peptide (ANP) and adrenomedullin (ADM) are potent hypotensive, diuretic, and natriuretic peptides involved in maintaining cardiovascular and renal homeostasis. We conducted a prospective 7-year study of 177 nondiabetic patients with primary chronic kidney disease to see if ANP and ADM plasma concentrations predict the progression of their disease, using novel sandwich immunoassays covering the midregional epitopes of the stable prohormones (MRproANP and MR-proADM). Progression of chronic kidney disease was defined as doubling of baseline serum creatinine and/or terminal renal failure, which occurred in 65 patients. Analysis of the receiver operating characteristic curve for the prediction of renal endpoints showed similar areas under the curve for the glomerular filtration rate (GFR) (0.838), MR-proANP (0.810), and MRproADM (0.876), respectively, as did the Kaplan–Meier curve analyses of the patients stratified according to the median of the respective markers. In separate multiple Cox-proportional hazard regression analyses, increased plasma concentrations of both peptides were each strongly predictive of the progression of chronic kidney disease after adjustments for age, gender, GFR, proteinuria and amino-terminal pro-B-type natriuretic peptide. Our study suggests that MR-proANP and MR-proADM are useful new markers of progression of primary nondiabetic chronic kidney disease.

High-density lipoprotein (HDL) is an attractive target for antiatherogenic drug therapy because of the inverse association between HDL cholesterol and cardiovascular risk as well as many potentially antiatherogenic functions. However, controversial data from inborn errors of human HDL metabolism and genetic animal models as well as the frequent confounding of low HDL cholesterol with other proatherogenic conditions in the population have complicated the proof of a causal relationship between HDL cholesterol and atherosclerosis. Because HDLs form a very heterogenous class of lipoproteins which differ in protein and lipid composition, it is increasingly accepted that the quality rather than quantity of HDL is relevant for its atheroprotective activity. As a consequence, protein or lipid or functional biomarkers are postulated to be better biomarkers than HDL cholesterol to assess and monitor the cardiovascular risk exerted by disturbed HDL metabolism and to estimate the benefit of any therapeutic intervention. In addition, novel therapeutics are searched which either improve HDL metabolism, mimic HDL function or cure the regulatory network underlying disturbed HDL metabolism and function. Beyond better biomarkers of HDL functionality and metabolism early clinical endpoint studies are needed to assess the therapeutic benefit of any novel HDL-modifying therapy.

Background: Individuals with severe deficiency in serum α1-antitrypsin (AAT) concentrations are at high risk for developing chronic obstructive pulmonary disease (COPD), whereas those carrying the PI*MZ genotype are at slightly increased risk. Testing appropriate subgroups of the population for AAT deficiency (AATD) is therefore an important aspect of COPD prevention and timely treatment. We decided to perform an exhaustive investigation of SERPINA1 gene variants in individuals from the general population with a moderately reduced serum AAT concentration, because such information is currently unavailable. Methods: We determined the Z and S alleles of 1399 individuals enrolled in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) with serum AAT concentrations ≤1.13 g/L and submitted 423 of these samples for complete exon 2→5 sequencing. Results: We found that 900 of 1399 samples (64%), carried the normal PI*MM genotype, whereas 499 samples (36%) carried at least 1 SERPINA1 deficiency variant. In the subpopulations in which AAT concentrations ranged from >1.03 to ≤1.13 and from >0.93 to ≤1.03 g/L, individuals with the PI*MM genotype represented the majority (86.5% and 53.8%, respectively). The PI*MS genotype was predominant (54.9%) in the AAT range of 0.83 to 0.93 g/L, whereas PI*MZ represented 76.4% in the AAT range of >0.73 to ≤0.83 g/L. Conclusions: This analysis provided a detailed molecular definition of intermediate AATD, which would be helpful in the diagnostic setting.

27-Hydroxycholesterol (27OH-Chol) is of potential diagnostic interest due to its role in maintaining whole-body cholesterol homeostasis. Dopant assisted-atmospheric pressure photoionization (DA-APPI) has improved the sensitivity of 27OH-Chol analysis, in comparison to the published LC-APCI-MS method, allowing quantification from a very low amount of sample (

Impaired endothelial function was demonstrated in HIV-infected persons on protease inhibitor (PI)-containing antiretroviral therapy, probably due to altered lipid metabolism. Atazanavir is a PI causing less atherogenic lipoprotein changes. This study determined whether endothelial function improves after switching from other PI to atazanavir.Randomised, observer-blind, treatment-controlled trial.Three university-based outpatient clinics.39 HIV-infected persons with suppressed viral replication on PI-containing regimens and fasting low-density lipoprotein (LDL)-cholesterol greater than 3 mmol/l.Patients were randomly assigned to continue the current PI or change to unboosted atazanavir.Endpoints at week 24 were endothelial function assessed by flow-mediated dilation (FMD) of the brachial artery, lipid profiles and serum inflammation and oxidative stress parameters.Baseline characteristics and mean FMD values of the two treatment groups were comparable (3.9% (SD 1.8) on atazanavir versus 4.0% (SD 1.5) in controls). After 24 weeks' treatment, FMD decreased to 3.3% (SD 1.4) and 3.4% (SD 1.7), respectively (all p = ns). Total cholesterol improved in both groups (p0.0001 and p = 0.01, respectively) but changes were more pronounced on atazanavir (p = 0.05, changes between groups). High-density lipoprotein and triglyceride levels improved on atazanavir (p = 0.03 and p = 0.003, respectively) but not in controls. Serum inflammatory and oxidative stress parameters did not change; oxidised LDL improved significantly in the atazanavir group.The switch from another PI to atazanavir in treatment-experienced patients did not result in improvement of endothelial function despite significantly improved serum lipids. Atherogenic lipid profiles and direct effects of antiretroviral drugs on the endothelium may affect vascular function.NCT00447070.

OBJECTIVE: Hyperhydration and exercise-associated hyponatremia (EAH) are critical issues during endurance events. We studied a cohort of marathon runners to examine EAH's prevalence in a marathon with a short time limit and to investigate underlying mechanisms that may be responsible for its development. DESIGN: Observational cohort study. SETTING: 2006 Zurich Marathon (cool and rainy weather, time limit of 5 hours). PARTICIPANTS: 167 marathon runners were recruited the month before the race. MAIN OUTCOME MEASURES: Body mass, plasma sodium, and osmolality were measured just before the start and immediately after the race. Fluid intake during the race was ascertained by a recall questionnaire. RESULTS: Five subjects (3 %) developed asymptomatic EAH, and no symptomatic EAH was found. Body mass change during the race correlated similarly with postrace sodium levels (r = -0.72, P < 0.0001) and with sodium change during the race (r = -0.66, P < 0.0001). Postrace sodium levels correlated significantly with sodium change during the race (r = 0.74, P < 0.0001). Fluid intake correlated significantly (r = -0.43, P < 0.0001) with plasma sodium change between the start and finish of the race. Postrace sodium levels and postrace osmolality were significantly correlated (r = 0.68, P < 0.0001). CONCLUSION: In this study we observed a relatively low incidence of EAH in subjects running the marathon in around 2.5 to 5 hours and in a cool environment. Plasma sodium change during the race and postrace sodium levels correlated with body mass change. There was also a direct correlation between fluid intake and plasma sodium change during the race.

Background Severe alpha1-antitrypsin (AAT) deficiency associated with low AAT blood concentrations is an established genetic COPD risk factor. Less is known about the respiratory health impact of variation in AAT serum concentrations in the general population. We cross-sectionally investigated correlates of circulating AAT concentrations and its association with FEV1. Methods In 5187 adults (2669 females) with high-sensitive c-reactive protein (CRP) levels ≤ 10 mg/l from the population-based Swiss SAPALDIA cohort, blood was collected at the time of follow-up examination for measuring serum AAT and CRP. Results Female gender, hormone intake, systolic blood pressure, age in men and in postmenopausal women, as well as active and passive smoking were positively, whereas alcohol intake and BMI inversely correlated with serum AAT levels, independent of CRP adjustment. We observed an inverse association of AAT with FEV1 in the total study population (p < 0.001), that disappeared after adjustment for CRP (p = 0.28). In addition, the AAT and FEV1 association was modified by gender, menopausal status in women, and smoking. Conclusion The results of this population-based study reflect a complex interrelationship between tobacco exposure, gender related factors, circulating AAT, systemic inflammatory status and lung function.

The ATP-binding cassette transporters ABCA1 and ABCG1 as well as scavenger receptor BI (SR-BI) mediate the efflux of lipids from macrophages to apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). We used RNA interference in RAW264.7 macrophages to study the interactions of ABCA1, ABCG1, and SR-BI with lipid-free apoA-I, native and reconstituted HDL with apoA-I:phosphatidylcholine ratios of either 1:40 (rHDL(1:40)) or 1:100 (rHDL(1:100)). Knock-down of ABCA1 inhibits the cellular binding at 4 degrees C of lipid-free apoA-I but not of HDL whereas suppression of ABCG1 or SR-BI reduces the binding of HDL but not lipid-free apoA-I. The degree of lipidation influences the interactions of rHDL with ABCG1 and SR-BI. Knock-down of ABCG1 inhibits more effectively the binding and cholesterol efflux capacities of lipid-poorer rHDL(1:40) whereas knock-down of SR-BI has a more profound effect on the binding and cholesterol efflux capacities of lipid-richer rHDL(1:100). Moreover, knock-down of ABCG1 but not SR-BI interferes with the association of lipid-free apoA-I during prolonged incubation at 37 degrees C. Finally, knock-down of ABCG1 inhibits the binding of initially lipid-free apoA-I which has been preconditioned by cells with high ABCA1 activity. The gained ability of initially lipid-free apoA-I to interact with ABCG1 is accompanied by its shift from electrophoretic pre-beta- to alpha-mobility. Taken together, these data suggest that the interaction of lipid-free apoA-I with ABCA1 generates a particle that immediately interacts with ABCG1 but not with SR-BI. Furthermore, the degree of lipidation influences the interaction of HDL with ABCG1 or SR-BI.

Accumulation of lipid-loaded macrophages (foam cells) within the vessel wall is an early hallmark of atherosclerosis. High-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) can efficiently promote cholesterol efflux from macrophages. Therefore, the interaction of HDL and apoA-I with macrophages appears to be important in the initial steps of reverse cholesterol transport, i.e. the transport of excess cholesterol from foam cells to the liver. However, although several cellular apoA-I and HDL receptors and transporters have been identified, it is as yet controversial how these interactions lead to cholesterol efflux from foam cells. In this study, we show that RAW264.7 macrophages bind HDL and apoA-I in a compatible manner. Furthermore, cell surface biotinylation experiments revealed that apoA-I but not HDL is specifically internalised. Binding of HDL to macrophages is decreased by reducing the expression of scavenger receptor BI (SR-BI) with cyclic adenosine monophosphate (cAMP), acetylated low-density lipoprotein (acLDL) or RNA interference. In contrast, apoA-I cell association and internalisation is modulated in parallel with ATP-binding cassette transporter A1 (ABCA1) expression which is altered by stimulating cells with cAMP and acLDL or expressing short hairpin RNA (shRNA) against ABCA1. Consistent with this, cell surface trapping of ABCA1 with cyclosporin A (CsA) results in increased apoA-I binding but reduced internalisation. Furthermore, blocking apoA-I uptake inhibits cholesterol efflux to apoA-I but not to HDL. Taken together, these data suggest that apoA-I- but not HDL-mediated cholesterol efflux may involve retroendocytosis.

Background— Low-density lipoprotein (LDL) uptake by monocyte-derived macrophages is a crucial step in foam cell formation and early atherosclerotic lesion. Increasing evidence supports the theory that activation of protein kinase Cβ (PKCβ) is involved in many mechanisms promoting atherosclerosis. Thus, we investigated whether inhibition of PKCβ prevents foam cell formation. Methods and Results— The differentiation of human primary monocytes or the monocytic THP-1 cell line into monocyte-derived macrophages was induced by phorbol 12-myristate 13-acetate (PMA; 0.1 mmol/L), a potent activator of PKC. Incubation of monocyte-derived macrophages with DiI-modified LDL (acetylated LDL and oxidized LDL, 10 μg/mL) led to lipoprotein uptake. Interestingly enough, the nonselective inhibitor of PKCβ 1 and PKCβ 2 , LY379196 (5×10 −7 to 10 −5 mol/L), blunted LDL uptake in monocyte-derived macrophages as shown by flow cytometry. Specific siRNA-mediated knockdown of PKCβ exerted a similar effect. Furthermore, PMA alone and in the presence of modified LDL induced scavenger receptor A mRNA and protein expression, which was abolished by LY379196. CGP53353, a selective inhibitor of PKCβ 2 , did not affect LDL uptake, nor did it prevent scavenger receptor A upregulation. Incubation of monocyte-derived macrophages with PMA/LDL increased PKCβ 1 phosphorylation at the Thr-642 residue, which was blunted by LY379196. However, the expression of CD68, a marker of activated macrophages, was not affected by LY379196. Moreover, LY379196 did not affect lipopolysaccharide-induced CD14 degradation, tumor necrosis factor-α release, or superoxide anion production, ruling out any effect of PKCβ inhibition on innate immunity. Conclusions— Nonspecific inhibition of PKCβ prevents LDL uptake in macrophages. These findings suggest that PKCβ inhibitors may represent a novel class of antiatherosclerotic drugs.

Summary Antibody phage display is a powerful tool for the generation of monoclonal antibodies against virtually any given antigen. Chickens are phylogenetically more distant from humans compared to other laboratory animals, such as mice and rats. Therefore, the use of chickens is especially beneficial when generating recombinant antibodies against human autoantigens, which are often highly conserved among mammals. Another advantage of using chickens in antibody phage display is that the preparation of single chain variable fragment (scFv) antibody libraries is faster and easier compared to preparing such libraries from other species, as only two primer sets are needed for amplification of the chicken variable heavy chain (VH) and variable light chain (VL) genes. In the present study we explored the possibility to immunize chickens with antigen cocktails for the generation of recombinant antibody fragments directed to a range of human autoantigens. Two pairs of chickens were immunized with two cocktails of seven recombinant autoantigenic proteins, libraries were prepared and panned on the individual proteins. The polyclonal chicken sera reacted strongly with most of the antigens used for immunization. By creating and screening single-chain variable fragment antibody phage display libraries, recombinant monoclonal antibody fragments were isolated successfully against the autoantigens annexin XI, centromere protein B, heat shock protein B3, DNA topoisomerase I, histidyl tRNA synthetase, Ro52, Ro60, Rpp30 and U1A. In conclusion, the immunization of only four chickens with two distinct pools of a total of 14 autoantigenic proteins allowed the isolation of scFvs against nine of these antigens.

We assessed the potential of dual-energy computed tomography (CT) for the differentiation between uric acid (UA)-containing and non-UA-containing urinary stones. Forty urinary stones of 16 different compositions in two sizes (andor=5 mm) were examined in an ex vivo model. Thirty stones consisted of pure calcium oxalate (whewellite or wheddellite), calcium phosphate (apatite, brushite, or vaterite), ammonium magnesium phosphate (struvite), UA, ammonium acid urate, ammonium phosphate, sodium hydrogen urate, or cystine, and ten stones were of mixed composition (UA-sodium hydrogen urate, whewellite-urate, wheddellite-urate, whewellite-brushite, or whewellite-brushite-struvite). Scans were performed using dual-source CT in a dual-energy mode with the tubes simultaneously operating at 80 and 140 kV. Two readers analysed the data with respect to stone attenuation at each energy level. The stones were classified as UA- or non-UA-containing using manual attenuation measurements and software analysis results. Sensitivity, specificity, PPV, and NPV were calculated using crystallographic stone analysis as the gold standard. Twenty-six out of 40 stones (65%) contained no UA; 14 stones (35%) contained UA. When compared with UA-containing stones, the differences in attenuation values at 80 and 140 kV were significantly (P0.001) higher in stones containing no UA. The software automatically mapped 39/40 stones (98%). Only one (2%) 2 mm UA-stone was missed. The software correctly classified all detected stones as UA- or non-UA-containing. The attenuation values of the missed stone were manually plotted into the analysis sheet which allowed for the correct classification of the stone (containing UA). Therefore, the sensitivity, specificity, PPV, and NPV for the detection of UA-containing stones was 100%. Ex vivo experience indicates that differentiation between UA- and non-UA-containing stones can be accurately performed using dual-source dual-energy CT.

BACKGROUND Ciprofloxacin is widely used to treat respiratory tract infections. Like other fluoroquinolones, ciprofloxacin has immunomodulatory effects; however, it is unknown whether these effects are beneficial in the setting of lung transplantation. We investigated potential immunomodulatory effects of ciprofloxacin in a model of posttransplant bronchiolitis obliterans. METHODS The heterotopic tracheal transplantation model in rats was used. Three groups received ciprofloxacin and underwent different immunosuppressive regimens of cyclosporine A, that is, no immunosuppression, insufficient immunosuppression, or low-dose immunosuppression. Three groups underwent the same immunosuppressive regimen but had no ciprofloxacin treatment. Tracheas were harvested after 21 days and examined with respect to histology and expression of selected cytokines. RESULTS The allografts of animals treated with ciprofloxacin showed less airway obliteration compared with allografts of untreated animals. When combined with low-dose immunosuppression ciprofloxacin showed beneficial effects in preventing airway obliteration and rejection of the respiratory epithelium. Cytokine gene expression of the allografts treated with ciprofloxacin was higher with respect to transforming growth factor-beta and equal with respect to tumor necrosis factor-alpha and interferon-gamma compared with controls. When applied in combination with cyclosporine A, ciprofloxacin lowered the expression of transforming growth factor-beta and tumor necrosis factor-alpha and increased interferon-gamma expression. CONCLUSION Ciprofloxacin attenuates airway rejection after tracheal transplantation. Genetic expression of mediators that are known to play an important role in mediating rejection in this model supports an immunomodulatory and antifibrotic role of ciprofloxacin. These findings suggest that further clinical studies are needed to investigate whether ciprofloxacin in addition to its bactericidal effect might be beneficial in the treatment of human posttransplant bronchiolitis obliterans.

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Background: Bronchial hyperresponsiveness (BHR) and variation in glutathione S-transferase (GST) genes have been associated with asthma risk. The relationship of these two risk factors with adult onset asthma in the general population was investigated. Methods: GSTP1 Ile105Val single nucleotide polymorphism and GSTM1 and GSTT1 gene deletion polymorphisms were genotyped in the population-representative SAPALDIA cohort. BHR was assessed at baseline by methacholine challenge and defined as a fall of ⩾20% in forced expiratory volume in 1 s. Independent effects of GST polymorphisms and BHR on new onset of asthma after 11 years of follow-up were estimated by multiple logistic regression analysis, adjusting for relevant baseline measures. Effect modification was assessed by including interaction terms in the model. Results: Among 4426 asthma-free participants at baseline, 14% had BHR. At follow-up, 3.3% reported new onset of physician-diagnosed asthma. BHR (p GSTP1 Ile105Val genotype (p = 0.005) were independently associated with incident asthma, but no association was seen for GSTT1 and GSTM1 gene deletion polymorphisms. Among subjects free of respiratory symptoms at baseline, the effect of BHR on the risk of physician-diagnosed asthma at follow-up was restricted to GSTP1 105 Ile/Ile carriers (OR 4.57, 95% CI 2.43 to 8.57 vs 1.40, 95% CI 0.58 to 3.39; p for interaction = 0.023). Conclusions: If confirmed by independent studies, our results suggest that GSTP1 Ile105Val genotype strongly determines the progression of BHR to physician-diagnosed asthma in the general population.

Chronic inflammation plays a major role in atherogenesis and understanding the role of inflammation and its resolution will offer novel approaches to interfere with atherogenesis. The 15(S)-lipoxygenase (15-LOX) plays a janus-role in inflammation with pro-inflammatory and anti-inflammatory effects in cell cultures and primary cells and even opposite effects on atherosclerosis in two different animal species. There is evidence for a pro-atherosclerotic effect of 15-LOX including the direct contribution to LDL oxidation and to the recruitment of monocytes to the vessel wall, its role in angiotensin II mediated mechanisms and in vascular smooth muscle cell proliferation. In contrast to the pro-atherosclerotic effects of 15-LOX, there is also a broad line of evidence that 15-LOX metabolites of arachidonic and linoleic acid have anti-inflammatory effects. The 15-LOX arachidonic acid metabolite 15-HETE inhibits superoxide production and polymorphonuclear neutrophil (PMN) migration across cytokine-activated endothelium and can be further metabolized to the anti-inflammatory lipoxins. These promote vasorelaxation in the aorta and counteract the action of most other pro-inflammatory factors like leukotrienes and prostanoids. Anti-atherogenic properties are also reported for the linoleic acid oxidation product 13-HODE through inhibition of adhesion of several blood cells to the endothelium. Furthermore, there is evidence that 15-LOX is involved in the metabolism of the long-chain omega-3 fatty acid docosahexaenoic acid (DHA) leading to a family of anti-inflammatory resolvins and protectins. From these cell culture and animal studies the role of the 15-LOX in human atherosclerosis cannot be predicted. However, recent genetic studies characterized the 15-LOX haplotypes in Caucasians and discovered a functional polymorphism in the human 15-LOX promoter. This will now allow large studies to investigate an association of 15-LOX with coronary artery disease and to answer the question whether 15-LOX is pro- or anti-atherogenic in humans.

Background: Automated analysis of insoluble urine components can reduce the workload of conventional microscopic examination of urine sediment and is possibly helpful for standardization. We compared the diagnostic performance of two automated urine sediment analyzers and combined dipstick/automated urine analysis with that of the traditional dipstick/microscopy algorithm. Methods: A total of 332 specimens were collected and analyzed for insoluble urine components by microscopy and automated analyzers, namely the Iris iQ200 (Iris Diagnostics) and the UF-100 flow cytometer (Sysmex). Results: The coefficients of variation for day-to-day quality control of the iQ200 and UF-100 analyzers were 6.5% and 5.5%, respectively, for red blood cells. We reached accuracy ranging from 68% (bacteria) to 97% (yeast) for the iQ200 and from 42% (bacteria) to 93% (yeast) for the UF-100. The combination of dipstick and automated urine sediment analysis increased the sensitivity of screening to approximately 98%. Conclusions: We conclude that automated urine sediment analysis is sufficiently precise and improves the workflow in a routine laboratory. In addition, it allows sediment analysis of all urine samples and thereby helps to detect pathological samples that would have been missed in the conventional two-step procedure according to the European guidelines. Although it is not a substitute for microscopic sediment examination, it can, when combined with dipstick testing, reduce the number of specimens submitted to microscopy. Visual microscopy is still required for some samples, namely, dysmorphic erythrocytes, yeasts, Trichomonas, oval fat bodies, differentiation of casts and certain crystals. Clin Chem Lab Med 2007;45:1251-6

AIMS: We investigated whether myeloid-related protein 8/14 complex (MRP8/14) expressed by infiltrating monocytes and granulocytes may represent a mediator and early biomarker of acute coronary syndromes (ACS). METHODS AND RESULTS: Immunohistochemistry of coronary thrombi was done in 41 ACS patients. Subsequently, levels of MRP8/14 were assessed systemically in 75 patients with ACS and culprit lesions, with stable coronary artery disease (CAD), or with normal coronary arteries. In a subset of patients, MRP8/14 was measured systemically and at the site of coronary occlusion. Macrophages and granulocytes, but not platelets stained positive for MRP8/14 in 76% of 41 thrombi patients. In ACS, local MRP8/14 levels [22.0 (16.2-41.5) mg/L] were increased when compared with systemic levels [13.4 (8.1-14.7) mg/L, P = 0.03]. Systemic levels of MRP8/14 were markedly elevated [15.1 (12.1-21.8) mg/L, P = 0.001] in ACS when compared with stable CAD [4.6 (3.5-7.1) mg/L] or normals [4.8 (4.0-6.3) mg/L]. Using a cut-off level of 8 mg/L, MRP8/14 but not myoglobin or troponin, identified ACS presenting within 3 h from symptom onset. CONCLUSION: In ACS, MRP8/14 is markedly expressed at the site of coronary occlusion by invading phagocytes. The occurrence of elevated MRP8/14 in the systemic circulation prior to markers of myocardial necrosis makes it a prime candidate for the detection of unstable plaques and management of ACS.

BACKGROUND: The clinical course of HIV-1 infection is highly variable among individuals, at least in part as a result of genetic polymorphisms in the host. Toll-like receptors (TLRs) have a key role in innate immunity and mutations in the genes encoding these receptors have been associated with increased or decreased susceptibility to infections. OBJECTIVES: To determine whether single-nucleotide polymorphisms (SNPs) in TLR2-4 and TLR7-9 influenced the natural course of HIV-1 infection. METHODS: Twenty-eight SNPs in TLRs were analysed in HAART-naive HIV-positive patients from the Swiss HIV Cohort Study. The SNPs were detected using Sequenom technology. Haplotypes were inferred using an expectation-maximization algorithm. The CD4 T cell decline was calculated using a least-squares regression. Patients with a rapid CD4 cell decline, less than the 15th percentile, were defined as rapid progressors. The risk of rapid progression associated with SNPs was estimated using a logistic regression model. Other candidate risk factors included age, sex and risk groups (heterosexual, homosexual and intravenous drug use). RESULTS: Two SNPs in TLR9 (1635A/G and +1174G/A) in linkage disequilibrium were associated with the rapid progressor phenotype: for 1635A/G, odds ratio (OR), 3.9 [95% confidence interval (CI),1.7-9.2] for GA versus AA and OR, 4.7 (95% CI,1.9-12.0) for GG versus AA (P = 0.0008). CONCLUSION: Rapid progression of HIV-1 infection was associated with TLR9 polymorphisms. Because of its potential implications for intervention strategies and vaccine developments, additional epidemiological and experimental studies are needed to confirm this association.

Background: Plasma concentrations of B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are diagnostic and prognostic biomarkers of heart failure and are also increased in patients with chronic kidney disease (CKD). We examined the relevance of BNP and NT-proBNP as predictors of CKD progression. Methods: Of 227 nondiabetic patients with mild-to-moderate renal insufficiency, 177 patients ages 18–65 years were followed in a prospective multicenter cohort study for a period of ≤7 years. CKD progression was assessed by recording renal endpoints, defined as doubling of baseline serum creatinine or end-stage renal disease (ESRD) requiring renal replacement therapy. Results: BNP and NT-proBNP were significantly higher among 65 patients who reached the combined renal endpoint than among the 112 who did not [median (interquartile range) 61 (27–98) ng/L vs 39 (20–70) ng/L, P = 0.023, for BNP; 320 (117–745) ng/L vs 84 (44–176) ng/L, P

Objectives Oxidative stress in plasma may be promoted by plasma thiols such as homocysteine. However, other thiols such as glutathione may also exert antioxidant effects in vitro and in vivo. To further investigate whether plasma thiols act as prooxidants or antioxidants, we compared plasma oxidative status in patients with coronary heart disease (CHD) and in subjects occupationally exposed to carbon disulfide (CS 2 ). Methods Fifty-five subjects chronically exposed to CS 2 , 53 CHD patients, and 52 healthy controls were examined. To assess plasma oxidative status, concentrations of thiobarbituric reactive substances (TBARS) and total antioxidative capacity (TAC), as well as ferritin and ceruloplasmin were determined. Antioxidative reserve was assessed by the determination of vitamine E, uric acid, superoxide dismutase, catalase, and glutathion peroxidase. In addition, protein and non-protein plasma thiol levels were measured. Results Patients in both groups had increased levels of plasma thiols as compared to controls: CS 2 -exposed subjects presented with increased levels of thiols associated with plasma proteins, whereas CHD patients presented with elevated total homocysteine and cysteine levels. TBARS were significantly increased and TAC was significantly decreased both in CS 2 -exposed subjects and in CHD patients. In addition decreased activity of glutathione peroxidase, an antioxidative enzyme inhibited by thiol-containing compounds, was noted in both groups. Conclusion These results demonstrate that regardless of their metabolic origin increased thiols are associated with increased oxidative stress in plasma.

Hepatic involvement is commonly observed in arenavirus infections, but the viral determinants of liver disease are only partially understood. Here we exploited newly developed reverse-genetic techniques withLymphocytic choriomeningitis virus(LCMV), the prototype arenavirus, to address specifically the contribution of the viral glycoprotein (GP) to liver pathogenicity. It is well established that strain WE, but not ARM, causes hepatitis in mice. We found that this property correlated with the superior capacity of WE to propagate in cultured macrophages and hepatocyte-derived cells. In mice, the ability to establish prolonged viraemia allowed the virus to propagate from initially infected Kupffer cells in the liver to neighbouring hepatocytes that underwent apoptosis. Reverse-genetic replacement of the GP in strain ARM with WE-GP resulted in only a very modest increase in liver pathogenicity, if any. Yet, an ARM-derived variant virus with a mutated polymerase gene caused severe liver disease when engineered to display WE-GP but considerably less when expressing ARM-GP. This reverse-genetic approach to an animal model of arenaviral hepatitis reveals a previously underestimated contributory role of the GP that alone is, however, insufficient to cause disease.

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66 Shc adaptor protein controls cellular responses to oxidative stress. Mice lacking p66 Shc (p66 Shc−/− ) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66 Shc−/− mouse. p66 Shc−/− and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66 Shc−/− and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endothelium-dependent relaxations, increased peroxynitrite (ONOO − ) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66 Shc−/− diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66 Shc−/− but not in WT mice. We report that p66 Shc−/− mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66 Shc adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.

24S- and 27-hydroxycholesterol are obligatory intermediates of cholesterol catabolism and play an important role in the maintenance of whole-body cholesterol homeostasis. Using an HPLC-MS method for oxysterol quantification, the distribution of esterified and unesterified oxysterols in lipoprotein subfractions as well as the influence of daytime, food intake and menstrual cycle on oxysterol concentrations were investigated in healthy volunteers. Moreover, reference intervals for 24S- and 27-hydroxycholesterol in plasma as well as the corresponding levels for 27-hydroxycholesterol in the HDL subfraction were established in 100 healthy volunteers. Both circulating oxysterols are mainly transported in association with HDL and LDL--primarily in the esterified form. No significant diurnal changes and no variations during menstrual cycle of either absolute or cholesterol-related plasma levels were detected. In contrast to 24S-hydroxycholesterol in plasma and 27-hydroxycholesterol in the HDL subfraction, the 95% reference intervals of 27-hydroxycholesterol both in plasma and the non-HDL subfraction were higher in males than in females. The concentrations of 27-hydroxycholesterol in plasma and the non-HDL subfraction showed strong positive correlations with the concentrations of cholesterol, non-HDL cholesterol and triglycerides. Our data on the lipoprotein distribution of oxysterols as well as on their intra- and inter-individual variation set the stage for future clinical studies.

BACKGROUND: Eye drops may contain phosphates as part of their buffer system. In the presence of epithelial keratopathy a high concentration of phosphate favours corneal calcification. To date European legislation does not require a quantitative declaration of the phosphates since buffers are regarded as additives. The knowledge of the phosphate concentration in medications helps to prevent corneal calcifications. Our study gives an overview on the amount of phosphate contained in antiglaucoma drops. METHODS: 21 samples of commercially available antiglaucoma drops were tested. The quantification of phosphate was performed using the molybdate method on a Modular P autoanalyzer. RESULTS: 10 of 21 (47 %) glaucoma drops had a phosphate concentration above physiological levels (> 1.45 mmol/L). A concentration higher than 100 mmol/L was found in four preparations that contained timolol. CONCLUSIONS: Many antiglaucoma drops contain unphysiological levels of phosphate, very high concentrations are found in some beta-blockers. These preparations have the potential to favour the formation of insoluble crystalline calcium phosphate deposits when used on a damaged corneal surface, and should therefore be avoided.