Citations

Abstract Background: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). Methods: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, 15N-labeled recombinant HSA (15NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides 42LVNEVTEFAK51 and 526QTALVELVK534 from albumin and 15NrHSA were referenced to the calibration curve. Results: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13–200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%–11.0% for the peptide 42LVNEVTEFAK51, and 1.9%–12.3% for the 526QTALVELVK534 peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%–14.8% for the 42LVNEVTEFAK51 peptide, and 6.4%–14.1% for the 526QTALVELVK534 peptide. For the 42LVNEVTEFAK51 peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R2 value of 0.97 and a regression line of y = 0.99x + 23.16. Conclusions: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a 15NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Background and Purpose— Size of an unruptured intracranial aneurysm (UIA) may be an important risk factor for rupture. Accordingly, serial noninvasive imaging is commonly used to assess untreated UIA for enlargement. Few data exist regarding the frequency and predictors of enlargement. We obtained this information from a group of patients followed with serial MR angiography (MRA). Methods— We retrospectively identified 165 patients with 191 UIA followed with serial MRA. Fusiform aneurysms, UIA <2 mm, and UIA that were surgically or endovascularly treated before the first MRA were excluded. MRA was performed using 1.5-T and 3-T MRI. Maximal diameter was determined on MRA source images. Multivariate regression analysis was used to determine independent risk factors for growth. Results— Twenty aneurysms (10%) grew over a median follow-up period of 47 months. Frequency of enlargement was 6.9%, 25%, and 83% for aneurysms <8 mm, 8 to 12 mm, and ≥13 mm, respectively ( P <0.001 for trend). Of the variables we evaluated, original aneurysm diameter (OR, 1.28 per mm; 95% CI, 1.07 to 1.58) was the only independent predictor of enlargement. Aneurysms ≥8 mm in diameter were at highest risk for enlargement (OR, 7.25; 95% CI, 1.96 to 27.1). There was a trend toward increased risk of enlargement in patients with multiple aneurysms (OR, 2.50; 95% CI, 0.86 to 7.53). Conclusions— Over a median follow-up period of 47 months, 10% of UIA enlarged. Larger aneurysms had a significantly increased risk of enlargement. The likelihood of enlargement was highest in aneurysms with diameters ≥8 mm. However, a clinically significant proportion of small aneurysms grow, and this growth can be detected by serial MRA.

Abstract Background: The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC. Methods: We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (&lt;20 mg/L, 20–250 mg/L, &gt;250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (Accumin™) assays. Results: The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations &lt;20 mg/L. Conclusions: Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.

Objective: To review the literature concerning the physiology of the endocannabinoid system, current drug development of cannabinoid agonists, and current clinical research on the use of cannabinoid agonists for analgesia. Data Sources: Articles were identified through a search of MEDLINE (1966–August 2005) using the key words cannabis, cannabinoid, cannabi*, cannabidiol, nabilone, THC, pain, and analgesia. No search limits were included. Additional references were located through review of the bibliographies of the articles identified. Study Selection and Data Extraction: Studies of cannabinoid agonists for treatment of pain were selected and were not limited by pain type or etiology. Studies or reviews using animal models of pain were also included. Articles that related to the physiology and pharmacology of the endocannabinoid system were evaluated. Data Synthesis: The discovery of cannabinoid receptors and endogenous ligands for these receptors has led to increased drug development of cannabinoid agonists. New cannabimimetic agents have been associated with fewer systemic adverse effects than delta-9-tetrahydrocannabinol, including recent development of cannabis medicinal extracts for sublingual use (approved in Canada), and have had promising results for analgesia in initial human trials. Several synthetic cannabinoids have also been studied in humans, including 2 cannabinoid agonists available on the international market. Conclusions: Cannabinoids provide a potential approach to pain management with a novel therapeutic target and mechanism. Chronic pain often requires a polypharmaceutical approach to management, and cannabinoids are a potential addition to the arsenal of treatment options.

Neurofibrillary tangles composed of hyperphosphorylated, aggregated tau are a common pathological feature of tauopathies, including Alzheimer's disease. Abnormal phosphorylation of tau by kinases or phosphatases has been proposed as a pathogenic mechanism in tangle formation. To investigate whether kinase inhibition can reduce tauopathy and the degeneration associated with it in vivo , transgenic mice overexpressing mutant human tau were treated with the glycogen synthase kinase-3 (GSK-3) inhibitor lithium chloride. Treatment resulted in significant inhibition of GSK-3 activity. Lithium administration also resulted in significantly lower levels of phosphorylation at several epitopes of tau known to be hyperphosphorylated in Alzheimer's disease and significantly reduced levels of aggregated, insoluble tau. Administration of a second GSK-3 inhibitor also correlated with reduced insoluble tau levels, supporting the idea that lithium exerts its effect through GSK-3 inhibition. Levels of aggregated tau correlated strongly with degree of axonal degeneration, and lithium-chloride-treated mice showed less degeneration if administration was started during early stages of tangle development. These results support the idea that kinases are involved in tauopathy progression and that kinase inhibitors may be effective therapeutically.

ABSTRACT Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

OBJECTIVES To examine the impact of time to thrombolytic treatment on multiple acute outcome variables in a single trial of thrombolysis in acute myocardial infarction. DESIGN AND PATIENTS Mortality and reinfarction rate were measured in 2770 patients with acute myocardial infarction who received thrombolysis within 12 hours in CORE, an international, dose ranging trial of poloxamer 188. Tc-99m sestamibi infarct size and radionuclide angiographic ejection fraction substudies included 1099 and 1074 patients, respectively. RESULTS Time to thrombolysis, subgrouped by intervals (< 2, 2–4, ⩾ 4–6, and ⩾ 6 hours), was significantly associated with infarct size (median 15.0%, 18.5%, 22.0%, 18.5% of left ventricle; p = 0.033), mean (SD) ejection fraction (51.5 (12.0)%, 48.3 (13.9)%, 48.2 (13.3)%, 48.2 (15.0)%; p = 0.006), 35 day mortality (5.7%, 7.1%, 7.9%, 12.5%; p = 0.0004), six month mortality (7.3%, 8.6%, 10.4%, 15.5%; p < 0.0001), and 35 day reinfarction rate (6.1%, 3.2%, 4.0%, 0.9%; p = 0.0001). CONCLUSIONS In this single large trial, the beneficial effect of time to thrombolysis on infarct size and ejection fraction was restricted to treatment given within two hours of symptom onset, while the effect on mortality was evident over all time intervals. Reinfarction rate was higher in patients treated with earlier thrombolysis.