Liberal fluid fasting: impact on gastric pH and residual volume in healthy children undergoing general anaesthesia for elective surgery.

BACKGROUND: The paediatric preoperative fasting time of 2 h for clear fluids, as suggested by guidelines, is often exceeded. Shorter preoperative fasting has been proposed to avoid potential outcomes such as dehydration, ketoacidosis, reduced arterial blood pressure, and patient discomfort. The aim of this study was to investigate whether liberal clear fluid intake until premedication significantly reduces actual fasting time and impacts gastric pH and residual volume. METHODS: Children (1-16 yr old, ASA I or II) undergoing elective procedures with general anaesthesia requiring tracheal intubation were randomised for clear fluid intake until premedication with midazolam (liberal) or 2 h fluid fasting (standard). Actual fasting times were recorded. Gastric content was sampled after tracheal intubation with an orogastric tube to determine gastric pH and residual volume. Data are presented as median [interquartile range]. RESULTS: We included 162 children aged 1.1-16 yr; gastric pH was determined in 138 patients. Patients' characteristics were similar in the two groups. The liberal fasting group had significantly shorter fasting times (48 [18.5-77.5] vs 234 [223.5-458.5] min; P<0.001). No significant difference was observed regarding gastric pH (1.6 [1.5-1.8] vs 1.6 [1.4-1.7]; P=0.237) or residual volume (0.38 [0.1-1.1] vs 0.43 [0.13-0.73] ml kg-1; P=0.535). Twelve patients (15%) in the liberal group (median fluid fasting 32 min) vs one patient (1%) had gastric residual volumes >2 ml kg-1 (P=0.001). CONCLUSION: Fluid intake until premedication allows for significantly shorter fasting times. Elevated gastric residual volumes may occur more often in patients with fasting times of 30 min or shorter. CLINICAL TRIAL REGISTRATION: NCT02603094.

Prospective long-term evaluation of parenteral hydroxocobalamin supplementation in juvenile beagles with selective intestinal cobalamin malabsorption (Imerslund-Gräsbeck syndrome).

BACKGROUND: Prospective studies on maintenance treatment for Beagles with hereditary selective cobalamin (Cbl) malabsorption (Imerslund-Gräsbeck syndrome, IGS) are lacking. In our experience, measurement of methylmalonic acid (MMA), a Cbl-dependent metabolite, seems more helpful to monitor Cbl status as compared with serum Cbl concentrations. OBJECTIVES: To evaluate a standardized Cbl supplementation scheme in Beagles with IGS. We hypothesized that a single parenteral dose of 1 mg hydroxocobalamin (OH-Cbl) would maintain clinical and metabolic remission for up to 2 months. ANIMALS: Six client-owned juvenile Beagles with genetically confirmed IGS and 28 healthy control dogs. METHODS: Prospective study. Monthly IM OH-Cbl (1 mg) supplementation was done over a median of 9 months (range, 6-13) in 6 dogs, followed by bimonthly (every 2 months) injections in 5 dogs over a median of 6 months (range, 3-10). Health status was assessed by routine clinical examinations at injection time points and owner observations. Voided urine samples were collected immediately before OH-Cbl injections for measurement of MMA-to-creatinine concentrations using a gas-liquid chromatography-tandem mass spectrometry (GC-MS) method. RESULTS: All dogs were clinically healthy while receiving monthly and bimonthly OH-Cbl supplementation. Urinary MMA results in healthy dogs ranged from 1.3 to 76.5 mmol/mol creatinine (median, 2.9). Median urinary MMA concentrations did not differ between dogs with IGS receiving monthly (n = 49; 5.3 mmol/mol creatinine; range, 2.3-50.4) and bimonthly (n = 31; 5.3 mmol/mol creatinine; range, 1.6-50) injections. CONCLUSIONS AND CLINICAL IMPORTANCE: A maintenance parenteral dose of 1 mg OH-Cbl monthly or bimonthly appears adequate in Beagles with IGS monitored by metabolic testing.

Effects of 6 Weeks of Parenteral Cobalamin Supplementation on Clinical and Biochemical Variables in Cats with Gastrointestinal Disease.

BACKGROUND: Effects and duration of commonly used protocols for cobalamin (Cbl) supplementation on cellular Cbl deficiency have not been determined in hypocobalaminemic cats. HYPOTHESIS/OBJECTIVES: To evaluate effect of Cbl supplementation on clinical signs, serum and urine methylmalonic acid (MMA) concentrations over 16 weeks. ANIMALS: Twenty client-owned hypocobalaminemic cats with enteropathy. METHODS: Prospective study. Serum Cbl and serum and urine MMA concentrations were determined prospectively in cats at enrollment (t0), immediately before (t6), and 4 (t10) and 10 weeks (t16) after 6th Cbl injection (250 µg, IM q 7 days). Clinical signs severity (activity, appetite, vomiting, diarrhea, body weight) graded at each time point and expressed as clinical disease activity score. RESULTS: Clinical disease activity score decreased during supplementation and increased after treatment discontinuation. Median serum Cbl concentration increased significantly from t0 (111 pmol/L, range 111-212) to t6 (2,332.5 pmol/L, range 123-22,730) (P < 0.01). Values at t10 were 610.5 pmol/L (range, 111-2,527) and 180.5 pmol/L (range, 111-2,262) at t16 (P < 0.01). Median baseline serum MMA concentration (372 µmol/L, range 0.39-147,000) decreased significantly to 1.62 µmol/L (range, 0.18-806) at t6 (P < 0.01) and gradually increased to 5.34 µmol/L (range, 0.13-1,730) at t10 and 189 µmol/L (range, 0.4-983) at t16. Similar, nonsignificant, pattern observed for urine MMA concentration. Serum and urine MMA concentrations had not normalized in 12 and 6 cats, respectively, at t6. CONCLUSION AND CLINICAL IMPORTANCE: The Cbl supplementation protocol used here did not lead to complete normalization of cellular Cbl deficiency in all examined cats, and biochemical improvements were transient.

Gender-Related Differences in Patients Presenting with Suspected Acute Coronary Syndromes: Clinical Presentation, Biomarkers and Diagnosis.

OBJECTIVES: Gender differences in patients presenting with suspected acute coronary syndromes (ACS) have not yet been fully characterized. The aim of this study was to assess gender-related disparities in clinical profiles, biomarkers and diagnoses of patients with suspected ACS. METHODS: This single-centre, prospective cohort study included 377 consecutive patients presenting with suspected ACS to the emergency department. Suspected ACS was defined as a request for conventional troponin T (c-cTnT) measurements on clinical grounds. RESULTS: Women were older than men (p = 0.004), and had a lower prevalence of known coronary artery and peripheral vascular disease (p < 0.05). c-cTnT was positive in 8% of female and in 14% of male patients (p = 0.16), TIMI risk score and cardiac biomarkers including c-cTnT, hs-cTnT, myoglobin, creatine kinase, N-terminal pro-brain natriuretic peptide, myeloid-related protein 8/14 and pregnancy-associated plasma protein A were lower in women (p < 0.05). Women were less frequently diagnosed with ACS (30 vs. 51%), and were not referred for urgent coronary angiography as often as men (p < 0.001). In multivariate analysis, female gender was associated with a lower referral for coronary angiography (HR 0.41, 95% CI 0.23-0.78, p = 0.006). CONCLUSIONS: In patients with suspected ACS, women presented with different biomarker profiles, and were less often diagnosed with ACS and referred to coronary angiography.

TLR9 agonists induced cell death in Burkitt's lymphoma cells is variable and influenced by TLR9 polymorphism.

Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt's lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity.

Enhanced glucose uptake via GLUT4 fuels recovery from calcium overload after ischaemia-reperfusion injury in sevoflurane- but not propofol-treated hearts.

BACKGROUND: So far, no study has explored the effects of sevoflurane, propofol, and Intralipid on metabolic flux rates of fatty acid oxidation (FOX) and glucose oxidation (GOX) in hearts exposed to ischaemia-reperfusion. METHODS: Isolated paced working rat hearts were exposed to 20 min of ischaemia and 30 min of reperfusion. Peri-ischaemic sevoflurane (2 vol%) and propofol (100 µM) in the formulation of 1% Diprivan(®) were assessed for their effects on oxidative energy metabolism and intracellular diastolic and systolic Ca(2+) concentrations. Substrate flux was measured using [(3)H]palmitate and [(14)C]glucose and [Ca(2+)] using indo-1AM. Western blotting was used to determine the expression of the sarcolemmal glucose transporter GLUT4 in lipid rafts. Biochemical analyses of nucleotides, ceramides, and 32 acylcarnitines were also performed. RESULTS: Sevoflurane, but not propofol, improved the recovery of left ventricular work (P=0.008) and myocardial efficiency (P=0.008) compared with untreated ischaemic hearts. This functional improvement was accompanied by reduced increases in post-ischaemic diastolic and systolic intracellular Ca(2+) concentrations (P=0.008). Sevoflurane, but not propofol, increased GOX (P=0.009) and decreased FOX (P=0.019) in hearts exposed to ischaemia-reperfusion. GLUT4 expression was markedly increased in lipid rafts of sevoflurane-treated hearts (P=0.016). Increased GOX closely correlated with reduced Ca(2+) overload. Intralipid alone decreased energy charge and increased long-chain and hydroxyacylcarnitine tissue levels, whereas sevoflurane decreased toxic ceramide formation. CONCLUSIONS: Enhanced glucose uptake via GLUT4 fuels recovery from Ca(2+) overload after ischaemia-reperfusion in sevoflurane- but not propofol-treated hearts. The use of a high propofol concentration (100 µM) did not result in similar protection.

Excellent agreement between genetic and hydrogen breath tests for lactase deficiency and the role of extended symptom assessment.

Clinical manifestations of lactase (LCT) deficiency include intestinal and extra-intestinal symptoms. Lactose hydrogen breath test (H2-BT) is considered the gold standard to evaluate LCT deficiency (LD). Recently, the single-nucleotide polymorphism C/T(-13910) has been associated with LD. The objectives of the present study were to evaluate the agreement between genetic testing of LCT C/T(-13910) and lactose H2-BT, and the diagnostic value of extended symptom assessment. Of the 201 patients included in the study, 194 (139 females; mean age 38, range 17-79 years, and 55 males, mean age 38, range 18-68 years) patients with clinical suspicion of LD underwent a 3-4 h H2-BT and genetic testing for LCT C/T(-13910). Patients rated five intestinal and four extra-intestinal symptoms during the H2-BT and then at home for the following 48 h. Declaring H2-BT as the gold standard, the CC(-13910) genotype had a sensitivity of 97% and a specificity of 95% with a κ of 0.9 in diagnosing LCT deficiency. Patients with LD had more intense intestinal symptoms 4 h following the lactose challenge included in the H2-BT. We found no difference in the intensity of extra-intestinal symptoms between patients with and without LD. Symptom assessment yielded differences for intestinal symptoms abdominal pain, bloating, borborygmi and diarrhoea between 120 min and 4 h after oral lactose challenge. Extra-intestinal symptoms (dizziness, headache and myalgia) and extension of symptom assessment up to 48 h did not consistently show different results. In conclusion, genetic testing has an excellent agreement with the standard lactose H2-BT, and it may replace breath testing for the diagnosis of LD. Extended symptom scores and assessment of extra-intestinal symptoms have limited diagnostic value in the evaluation of LD.

HMOX1 and GST variants modify attenuation of FEF25-75% decline due to PM10 reduction.

Reduced exposure to particulate matter with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)) attenuated age-related lung function decline in our cohort, particularly in the small airways. We hypothesised that polymorphisms in glutathione S-transferase (GST) and haem oxygenase-1 (HMOX1) genes, important for oxidative stress defence, modify these beneficial effects. A population-based sample of 4,365 adults was followed up after 11 yrs, including questionnaire, spirometry and DNA blood sampling. PM(10) exposure was estimated by dispersion modelling and temporal interpolation. The main effects on annual decline in forced expiratory flow at 25-75% of forced vital capacity (FEF(25-75%)) and interactions with PM(10) reduction were investigated for polymorphisms HMOX1 rs2071746 (T/A), rs735266 (T/A) and rs5995098 (G/C), HMOX1 (GT)(n) promoter repeat, GSTM1 and GSTT1 deletions, and GSTP1 p.Ile105Val, using mixed linear regression models. HMOX1 rs5995098, HMOX1 haplotype TTG and GSTP1 showed significant genetic main effects. Interactions with PM(10) reduction were detected: a 10 microg.m(-3) reduction significantly attenuated annual FEF(25-75%) decline by 15.3 mL.s(-1) only in the absence of HMOX1 haplotype ATC. Similarly, carriers of long (GT)(n) promoter repeat alleles or the GSTP1 Val/Val genotype profited significantly more from a 10 microg.m(-3) reduction (26.5 mL.s(-1) and 27.3 mL.s(-1) respectively) than non-carriers. Benefits of a reduction in PM(10) exposure are not equally distributed across the population but are modified by the individual genetic make-up determining oxidative stress defence.

Increased T-bet to GATA-3 ratio during acute allograft rejection in the rat lung.

Acute allograft rejection (AR) remains a major problem in solid organ transplantation. The pivotal mechanism hinges on alloantigen recognition by recipient T helper (T(h)) cells that differentiate into T(h)1 and T(h)2. This study investigated the association of mRNA levels of the transcription factors T-box expressed in T cells and GATA-binding protein 3 with the development of T(h)1/T(h)2-directed immune responses. We investigated the expression of T-bet and GATA-3 mRNA levels and the protein levels of their marker cytokines interleukin (IL)-2 and IL-4 in orthotopically transplanted rat lungs during AR. We observed a nonsignificant increase in T-bet expression following allografting at days 3 and 5 but there was a significant reduction in GATA-3 expression on day 5 compared with controls. The ratio of T-bet to GATA-3 expression showed a trend to increase at day 3 following allografting reaching significance at 5 days. These changes were associated with a significant increase in the expression of IL-2 over IL-4 on days 3 and 5. This study suggests that T(h)1 responses play a major role during AR in the rat lung, and that this differentiation can be monitored by measuring mRNA levels of T-bet and GATA-3.

Atherogenic forms of dyslipidaemia in women with polycystic ovary syndrome.

OBJECTIVE: Dyslipidaemia is very common in patients with polycystic ovary syndrome (PCOS) but, beyond plasma lipids, atherogenic lipoprotein (Lp) and apolipoprotein (apo) alterations are still ill defined. DESIGN: We measured concentrations of apoB, Lp(a) and small, dense low-density lipoprotein (LDL) in 42 patients with PCOS [age: 28 +/- 7 years, body mass index (BMI): 27 +/- 5 kg/m(2)] vs. 37 age- and BMI-matched healthy controls. METHODS: Elevated Lp(a) levels considered were those > 30 mg/dl while elevated apoB concentrations were those > 100 g/l. RESULTS: Polycystic ovary syndrome showed increased triglycerides levels (p = 0.0011) and lower high-density lipoprotein (HDL)-cholesterol concentrations (p = 0.0131) while total- and LDL cholesterol were similar. PCOS also showed smaller LDL size (p = 0.0005), higher levels of total small, dense LDL (p < 0.0001), higher concentrations of Lp(a), as considered as absolute values (p = 0.0143) and log-transformed (p = 0.0014), while no differences were found in apoB levels. Elevated Lp(a) concentrations were found in 24% of PCOS, while elevated apoB levels were relatively uncommon (14%). Spearman correlation analysis revealed that Lp(a) concentrations were weakly correlated only with HDL-cholesterol levels (r = -0.378, p = 0.0431). In addition, 36% of patients with PCOS with normal plasma lipid profile showed elevated levels of Lp(a), apoB or small, dense LDL. CONCLUSIONS: Atherogenic Lp abnormalities may be found in one-third of women with PCOS who have a normal lipid pattern. Future prospective studies are needed to test to which extent such atherogenic forms of dyslipidaemia may contribute to the increased cardiovascular risk in young women with PCOS.

Effect of atazanavir versus other protease inhibitor-containing antiretroviral therapy on endothelial function in HIV-infected persons: randomised controlled trial.

OBJECTIVE: 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. DESIGN: Randomised, observer-blind, treatment-controlled trial. SETTING: Three university-based outpatient clinics. PATIENTS: 39 HIV-infected persons with suppressed viral replication on PI-containing regimens and fasting low-density lipoprotein (LDL)-cholesterol greater than 3 mmol/l. INTERVENTION: Patients were randomly assigned to continue the current PI or change to unboosted atazanavir. MAIN OUTCOME MEASURES: 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. RESULTS: 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 (p<0.0001 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. CONCLUSIONS: 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. TRIAL REGISTRATION NUMBER: NCT00447070.

Pharmacokinetics and pharmacodynamics of cinacalcet in patients with hyperparathyroidism after renal transplantation.

Cinacalcet is a calcimimetic drug for the treatment of secondary hyperparathyroidism (HPT). In a sequential open-label study, ten patients with persistent HPT after renal transplantation received first 30 and then 60 mg oral cinacalcet once daily over 2 weeks each. Cinacalcet steady state oral clearance was 131.1 +/- 20.9 l/h and 92.8 +/- 9.5 l/h (mean +/- SE) after 30 and 60 mg, respectively. Cinacalcet and parathyroid hormone (PTH) concentrations showed an inverse correlation and were fitted to a simple E(max) model (E(max) = 80% reduction vs. baseline, EC(50) = 13 ng/mL). A once daily administration of cinacalcet lowered serum calcium over 24 h without fluctuations. The 8-h fractional urinary excretion of calcium was increased after 60 mg cinacalcet (baseline 0.85 +/- 0.17%, 30 mg 1.53 +/- 0.35%, 60 mg 1.92 +/- 0.37%). Renal function remained stable. Cinacalcet pharmacokinetics and pharmacodynamics showed a pronounced interindividual variability. We conclude that the once daily administration of cinacalcet in patients with secondary HPT after renal transplantation effectively reduced iPTH and serum calcium. The transient calciuria could potentially favor nephrocalcinosis and reduce bone mineral density, suggesting that higher doses of cinacalcet need to be used with caution in renal transplant recipients with severe persistent hyperparathyroidism.

TNF-alpha -308G>A polymorphism modulates cytokine serum concentrations and macrovascular complications in diabetic patients on aspirin.

BACKGROUND: Tumor necrosis factor (TNF)-alpha has pleiotropic effects in cytokine-mediated inflammation underlying atherogenesis. Activation of this inflammatory process is assumed to be different in diabetic and non-diabetic individuals. Previous studies in non-diabetic subjects showed no association between TNF-alpha -308G>A polymorphism and coronary artery disease. METHODS: Vascular complications and cytokine serum concentrations were assessed as a function of the TNF-alpha -308G>A polymorphism in 76 diabetic patients on low-dose aspirin. RESULTS: Of 76 adult diabetic patients, 18 (24%) carried the TNF-alpha -308A allele (17 AG, 1 AA) and 58 (76%) carried wild-type alleles (GG). Prevalence of macrovascular complications was 33% in TNF-alpha -308A allele carriers (AG+AA) and 78% in wild-type allele carriers (GG) (p<0.001). In contrast, prevalence of microvascular complications was 78% and 84%, respectively, and did not significantly differ between the study groups. TNF-alpha -308A allele carriers (AG+AA) compared to wild-type allele carriers (GG) had significantly lower median serum concentrations of hs-C-reactive protein (1.5 vs 2.9 mg/L, p=0.030), interleukin 1-beta (0.9 vs 1.2 ng/L, p=0.046), and interleukin-6 (3.6 vs 4.9 ng/L, p=0.023). In multiple regression analysis, the prevalence of macrovascular diabetic complications was significantly associated with TNF-alpha -308G>A polymorphism (p<0.001) and serum concentrations of HDL-cholesterol (p=0.007) while confounding effects of further variables were excluded. CONCLUSION: TNF-alpha -308G>A polymorphism modulates cytokine serum concentrations and macrovascular complications in diabetic patients on aspirin. Diabetic carriers of the TNF-alpha -308A allele might benefit more from a prophylaxis with low dose aspirin than non-carriers.

Hypocretin-1 (orexin A) deficiency in acute traumatic brain injury.

Hypocretin-1 is involved in the regulation of the sleep-wake cycle. The authors prospectively assessed CSF hypocretin-1 levels in 44 consecutive patients with acute traumatic brain injury (TBI). Compared with controls, hypocretin-1 levels were abnormally lower in 95% of patients with moderate to severe TBI and in 97% of patients with posttraumatic brain CT changes. Hypocretin-1 deficiency after TBI may reflect hypothalamic damage and be linked with the frequent development of posttraumatic sleep-wake disorders.

Modulation of high-density lipoprotein cholesterol metabolism and reverse cholesterol transport.

Low high-density lipoprotein (HDL)-cholesterol (C) is an important risk factor for coronary heart disease. In vitro, HDL exerts several potentially anti-atherogenic effects including reverse cholesterol transport (RCT) from peripheral cells to the liver. Hence, raising HDL-C has become an interesting target for anti-atherosclerotic drug therapy. Levels of HDL-C and the composition of HDL subclasses in plasma are regulated by apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters. The interplay of these factors leads to RCT and determines the composition and thereby the anti-atherogenic properties of HDL. Recent findings suggest that the mechanism of HDL modification rather than a sole increase in HDL-C determines the efficacy of anti-atherosclerotic drug therapy. In several controlled and prospective intervention studies, patients with low HDL-C and additional risk factors benefited from treatment with fibrates or statins. However, in only some of the fibrate trials was prevention of coronary events in patients with low HDL-C and hypertriglyceridaemia related to an increase in HDL-C. This may be because currently available drugs increase HDL-C levels only moderately and because HDL levels per se do not necessarily correlate with the functionality of HDL. However, several novel targets to modify RCT have emerged from the recent understanding of HDL synthesis, maturation and catabolism. The four major targets for an anti-atherogenic strategy in HDL metabolism include stimulation of apoA-I synthesis and secretion, the stimulation of ABCA1 expression, the inhibition of cholesterol ester transfer protein, and the up-regulation of scavenger receptor BI. These and other modulations of HDL metabolism are thought to result in improved RCT making them attractive targets for the development of new regimens of anti-atherogenic drug therapy.

CCTTT-repeat polymorphism of the inducible nitric oxide synthase is not associated with HIV pathogenesis.

Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis.

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures.

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full-length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1-eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP+1-eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1-eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.