Lysyl oxidase-like 2 as a predictor of hepatocellular carcinoma in patients with hepatitis C virus after sustained virological response.

Lysyl oxidase-like 2 (LOXL2) mediates the crosslinking of extracellular collagen, reflecting qualitative changes in liver fibrosis. This study aimed to validate the utility of serum LOXL2 levels as a predictive biomarker for the development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV) infection who achieved a sustained virological response (SVR). This retrospective study included 137 patients with chronic HCV infection without history of HCC development and who achieved SVR via direct-acting antiviral therapy. Median LOXL2 levels decreased significantly after SVR achievement (pre-Tx, 2.33 ng/mL; post-Tx, 1.31 ng/mL, p < 0.001). Post-Tx LOXL2 levels, fibrosis-4 index, platelet counts, Wisteria floribunda agglutinin-positive human Mac-2 binding protein levels, and alpha-fetoprotein (AFP) levels were identified as independent predictive factors for post-SVR HCC development in the univariate analysis. The incidence of post-SVR HCC development was significantly higher in patients with post-Tx LOXL2 levels ≥ 2.08 ng/mL and AFP levels ≥ 5.0 ng/mL than in patients with elevated levels of either marker or with lower marker levels. Serum LOXL2 levels can serve as a predictive biomarker for HCC development after achieving SVR. The combination of serum LOXL2 and AFP levels provides robust risk stratification for HCC development after SVR, suggesting an enhanced surveillance strategy.

Identification of potential biomarkers for predicting the early onset of diabetic cardiomyopathy in a mouse model.

Type 2 diabetes (T2D) is characterized by metabolic derangements that cause a shift in substrate preference, inducing cardiac interstitial fibrosis. Interstitial fibrosis plays a key role in aggravating left ventricular diastolic dysfunction (LVDD), which has previously been associated with the asymptomatic onset of heart failure. The latter is responsible for 80% of deaths among diabetic patients and has been termed diabetic cardiomyopathy (DCM). Through in silico prediction and subsequent detection in a leptin receptor-deficient db/db mice model (db/db), we confirmed the presence of previously identified potential biomarkers to detect the early onset of DCM. Differential expression of Lysyl Oxidase Like 2 (LOXL2) and Electron Transfer Flavoprotein Beta Subunit (ETFß), in both serum and heart tissue of 6-16-week-old db/db mice, correlated with a reduced left-ventricular diastolic dysfunction as assessed by high-resolution Doppler echocardiography. Principal component analysis of the combined biomarkers, LOXL2 and ETFß, further displayed a significant difference between wild type and db/db mice from as early as 9 weeks of age. Knockdown in H9c2 cells, utilising siRNA of either LOXL2 or ETFß, revealed a decrease in the expression of Collagen Type I Alpha1 (COL1A1), a marker known to contribute to enhanced myocardial fibrosis. Additionally, receiver-operating curve (ROC) analysis of the proposed diagnostic profile showed that the combination of LOXL2 and ETFß resulted in an area under the curve (AUC) of 0.813, with a cut-off point of 0.824, thus suggesting the favorable positive predictive power of the model and further supporting the use of LOXL2 and ETFß as possible early predictive DCM biomarkers.

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

Adiponectin and the steatosis marker Chi3L1 decrease following switch to raltegravir compared to continued PI/NNRTI-based antiretroviral therapy.

BACKGROUND: People with HIV are at for metabolic syndrome (MetS) and fatty liver disease, but the role of Antiretroviral therapy (ART) is poorly understood. MetS and fatty liver disease been associated with changes in adiponectin, soluble ST2 (sST2), chitinase 3-like 1 (Chi3L1), hyaluronic acid (HA), tissue inhibitor of metalloproteinase-1 (TIMP-1), lysyl oxidase-like-2 (LOXL2) and transforming growth factor ß (TGF-ß) concentrations in HIV-uninfected populations. Protease (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) may contribute to these comorbidities, but the effects of switching from PI- or NNRTI to raltegravir (RAL) on these biomarkers is unknown. METHODS: Cryopreserved plasma was obtained from a completed, prospective trial of HIV-infected women with central adiposity on NNRTI- or PI-based ART during which they were randomized to remain on their current ART or switch to a RAL based regimen. Biomarker concentrations were quantified using ELISA and Multiplex assays at baseline and 24 weeks after randomization. Wilcoxon-signed rank test evaluated within-group changes, Spearman and linear regression models evaluated correlations between biomarkers and clinical covariates. RESULTS: Participants had a median age of 43 years, CD4+ T lymphocyte count 558 cells/mm3 and BMI 32 kg/m2; 35% met criteria for MetS. At baseline, higher adiponectin levels correlated with higher Chi3L1 levels (r = 0.42, p = 0.02), as did declines after 24 weeks (r = 0.40, p = 0.03). Changes in sST2 correlated with changes in Chi3L1 (r = 0.43, p = 0.02) and adiponectin (r = 0.40, p = 0.03). Adiponectin and Chi3L1 levels decreased significantly in women switched to RAL vs continue PI/NNRTI. CONCLUSION: In women with HIV and central obesity, the hepatic steatosis/fibrosis marker Chi3L1 and adiponectin decrease in conjunction with sST2 decreases following switch to RAL. Whether switching from NNRTI/PI-based regimens to RAL can improve hepatic steatosis and dysmetabolism requires further study. TRIAL REGISTRATION: Clinicaltrials.gov NCT00656175.

The serum level and significance of lysyl oxidase-like 2 in patients with rheumatoid arthritis-associated interstitial lung disease.

Our previous experiments found that lysyl oxidase-like 2 (LOXL2) may be a useful preclinical serological marker for pulmonary fibrosis in the mouse model. The role of LOXL2 in rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is still unclear. We investigated whether serum LOXL2 levels are associated with RA-ILD patients. The levels of serum LOXL2 were measured by enzyme-linked immunosorbent assay in 49 RA-ILD patients (21 patients with ILD disease duration < 3 months; 28 patients with ILD disease duration > 3 months), 43 RA patients without ILD and 20 normal healthy controls. We assessed the correlations between the serum LOXL2 levels and clinical variables. Serum LOXL2 levels were significantly higher in RA patients than in normal healthy controls (326.79 ± 192.56 vs. 53.27 ± 35.86 pg/ml, P < 0.01). No significant difference was present between the RA-ILD group and RA without ILD group (298.87 ± 219.85 vs. 358.60 ± 152.16 pg/ml, P = 0.13). Notably, the serum LOXL2 levels were significantly higher in patients with ILD disease duration < 3 months than in those with ILD disease duration > 3 months (462.71 ± 208.97 vs. 175.99 ± 130.55 pg/ml, P < 0.01) or without ILD (462.71 ± 208.97 vs. 358.60 ± 152.16 pg/ml, P = 0.03). The serum LOXL2 levels in RA-ILD patients significantly correlated with DAS28 (rs = 0.31, P = 0.034), C-reactive protein (rs = 0.41, P = 0.004), rheumatoid factor (rs = 0.41, P = 0.003), forced vital capacity (rs = - 0.39, P = 0.02), and diffusion capacity of the lung for carbon monoxide (rs = - 0.44, P = 0.009). LOXL2 may be involved in the pathogenesis of rheumatoid arthritis-associated interstitial lung disease and might be helpful in early diagnosis of RA-ILD.

Increased serum lysyl oxidase-like 2 levels correlate with the degree of left atrial fibrosis in patients with atrial fibrillation.

Atrial fibrillation (AF) progression is generally accompanied by increased atrial fibrosis and atrial structural remodeling. Lysyl oxidase-like 2 (LOXL2) is known to play an important role in many fibrotic conditions, including cardiac fibrosis. The present study aimed to explore the relationship between serum LOXL2 levels and AF. Fifty-four AF patients and 32 control subjects were enrolled in the study. High-density three-dimensional electroanatomic mapping was performed, and mean bipolar voltage was assessed in AF patients. LOXL2 levels were measured by enzyme-linked immunosorbent assay. All patients underwent echocardiography to assess left atrium size and left ventricle function. Serum LOXL2 levels were significantly elevated in AF patients compared with the control group (526.81 ± 316.82 vs 240.94 ± 92.51 pg/ml, P<0.01). In addition, serum LOXL2 level was significantly correlated with the size of the left atrium (LAD) (r2 = 0.38, P<0.01). Furthermore, the serum LOXL2 levels were significantly higher in AF patients with LAD ≥ 40 mm compared with those with LAD < 40 mm (664.34 ± 346.50 vs 354.90 ± 156.23 pg/ml, P<0.01). And the Spearman's correlation analysis further revealed that the mean bipolar left atrial voltage was inversely correlated with the LOXL2 (r2 = -0.49, P<0.01) in AF patients. Multivariate regression analysis further demonstrated that serum LOXL2 [odds ratio (OR) 1.013, 95% confidence interval (CI) 1.002-1.024, P<0.05] and LAD (OR 1.704, 95% CI 1.131-2.568, P<0.01) were independent predictors of AF. In conclusion, serum LOXL2 levels were significantly elevated and were correlated with the degree of left atrial fibrosis in AF patients.

Influencing the Tumor Microenvironment: A Phase II Study of Copper Depletion Using Tetrathiomolybdate in Patients with Breast Cancer at High Risk for Recurrence and in Preclinical Models of Lung Metastases.

PURPOSE: Bone marrow-derived progenitor cells, including VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent pathways, model the tumor microenvironment. We hypothesized that copper depletion using tetrathiomolybdate would reduce EPCs in high risk for patients with breast cancer who have relapsed. We investigated the effect of tetrathiomolybdate on the tumor microenvironment in preclinical models. EXPERIMENTAL DESIGN: Patients with stage II triple-negative breast cancer (TNBC), stage III and stage IV without any evidence of disease (NED), received oral tetrathiomolybdate to maintain ceruloplasmin (Cp) between 8 and 17 mg/dL for 2 years or until relapse. Endpoints were effect on EPCs and other biomarkers, safety, event-free (EFS), and overall survival (OS). For laboratory studies, MDA-LM2-luciferase cells were implanted into CB17-SCID mice and treated with tetrathiomolybdate or water. Tumor progression was quantified by bioluminescence imaging (BLI), copper depletion status by Cp oxidase levels, lysyl oxidase (LOX) activity by ELISA, and collagen deposition. RESULTS: Seventy-five patients enrolled; 51 patients completed 2 years (1,396 cycles). Most common grade 3/4 toxicity was neutropenia (3.7%). Lower Cp levels correlated with reduced EPCs (P = 0.002) and LOXL-2 (P < 0.001). Two-year EFS for patients with stage II-III and stage IV NED was 91% and 67%, respectively. For patients with TNBC, EFS was 90% (adjuvant patients) and 69% (stage IV NED patients) at a median follow-up of 6.3 years, respectively. In preclinical models, tetrathiomolybdate decreased metastases to lungs (P = 0.04), LOX activity (P = 0.03), and collagen crosslinking (P = 0.012). CONCLUSIONS: Tetrathiomolybdate is safe, well tolerated, and affects copper-dependent components of the tumor microenvironment. Biomarker-driven clinical trials in high risk for patients with recurrent breast cancer are warranted. Clin Cancer Res; 23(3); 666-76. ©2016 AACR.

Efficacy of simtuzumab versus placebo in patients with idiopathic pulmonary fibrosis: a randomised, double-blind, controlled, phase 2 trial.

BACKGROUND: Lysyl oxidase-like 2 (LOXL2) catalyses collagen cross-linking and is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim of this study was to investigate the efficacy and safety of simtuzumab, a monoclonal antibody against LOXL2, in patients with IPF. METHODS: In this randomised, double-blind, phase 2 trial, we recruited patients aged 45-85 years with definite IPF diagnosed prior to 3 years of screening from 183 hospitals and respiratory clinics in 14 countries. Eligible patients, stratified by baseline forced vital capacity (FVC), serum LOXL2 (sLOXL2) concentrations, and pirfenidone and nintedanib use, were randomly assigned (1:1) to inject 125 mg/mL simtuzumab or placebo subcutaneously once a week. The primary endpoints were progression-free survival, defined as time to all-cause death or a categorical decrease from baseline in FVC % predicted, in the intention-to-treat population, in patients with sLOXL2 concentrations in the 50th percentile or higher, and in patients with sLOXL2 concentrations in the 75th percentile or higher. Treatment duration was event-driven, and interim analyses were planned and conducted after approximately 120 and 200 progression-free survival events, respectively, occurred. We compared treatment groups with the stratified log-rank test. This study is registered with ClinicalTrials.gov, number NCT01769196. FINDINGS: Patients with IPF were recruited between Jan 31, 2013, and June 1, 2015. The intention-to-treat population included 544 randomly assigned patients (272 patients in both groups), and the safety population included 543 randomly assigned patients who received at least one dose of study medication. The study was terminated when the second interim analysis met the prespecified futility stopping criteria in the intention-to-treat population. We noted no difference in progression-free survival between simtuzumab and placebo in the intention-to-treat population (median progression free survival times of 12·6 months and 15·4 months for simtuzumab and placebo, respectively; stratified HR 1·13, 95% CI 0·88-1·45; p=0·329) and in patients with baseline sLOXL2 in the 50th percentile or higher (median progression-free survival 11·7 months and 14·3 months for simtuzumab and placebo, respectively; stratified HR 1·03, 95% CI 0·74-1·43; p=0·851), or in the 75th percentile or higher (median progression-free survival 11·6 months and 16·9 months for simtuzumab and placebo, respectively; stratified HR 1·20, 95% CI 0·72-2·00; p=0·475). The incidence of adverse events and serious adverse events was similar between treatment groups. The most common adverse events in both the simtuzumab and placebo groups were dyspnoea, cough, upper respiratory tract infection, and worsening of IPF; and the most common grade 3 or 4 adverse events were worsening of IPF, dyspnoea, and pneumonia. INTERPRETATION: Simtuzumab did not improve progression-free survival in a well-defined population of patients with IPF. Our data do not support the use of simtuzumab for patients with IPF. FUNDING: Gilead Sciences Inc.

Targeting LOXL2 for cardiac interstitial fibrosis and heart failure treatment.

Interstitial fibrosis plays a key role in the development and progression of heart failure. Here, we show that an enzyme that crosslinks collagen-Lysyl oxidase-like 2 (Loxl2)-is essential for interstitial fibrosis and mechanical dysfunction of pathologically stressed hearts. In mice, cardiac stress activates fibroblasts to express and secrete Loxl2 into the interstitium, triggering fibrosis, systolic and diastolic dysfunction of stressed hearts. Antibody-mediated inhibition or genetic disruption of Loxl2 greatly reduces stress-induced cardiac fibrosis and chamber dilatation, improving systolic and diastolic functions. Loxl2 stimulates cardiac fibroblasts through PI3K/AKT to produce TGF-ß2, promoting fibroblast-to-myofibroblast transformation; Loxl2 also acts downstream of TGF-ß2 to stimulate myofibroblast migration. In diseased human hearts, LOXL2 is upregulated in cardiac interstitium; its levels correlate with collagen crosslinking and cardiac dysfunction. LOXL2 is also elevated in the serum of heart failure (HF) patients, correlating with other HF biomarkers, suggesting a conserved LOXL2-mediated mechanism of human HF.

New frontiers in fibrotic disease therapies: The focus of the Joan and Joel Rosenbloom Center for Fibrotic Diseases at Thomas Jefferson University.

Fibrotic diseases constitute a world-wide major health problem, but research support remains inadequate in comparison to the need. Although considerable understanding of the pathogenesis of fibrotic reactions has been attained, no completely effective therapies exist. Although fibrotic disorders are diverse, it is universally appreciated that a particular cell type with unique characteristics, the myofibroblast, is responsible for replacement of functioning tissue with non-functional scar tissue. Understanding the cellular and molecular mechanisms responsible for the creation of myofibroblasts and their activities is central to the development of therapies. Critical signaling cascades, initiated primarily by TGF-ß, but also involving other cytokines which stimulate pro-fibrotic reactions in the myofibroblast, offer potential therapeutic targets. However, because of the multiplicity and complex interactions of these signaling pathways, it is very unlikely that any single drug will be successful in modifying a major fibrotic disease. Therefore, we have chosen to examine the effectiveness of administration of several drug combinations in a mouse pneumoconiosis model. Such treatment proved to be effective. Because fibrotic diseases that tend to be chronic, are difficult to monitor, and are patient variable, implementation of clinical trials is difficult and expensive. Therefore, we have made efforts to identify and validate non-invasive biomarkers found in urine and blood. We describe the potential utility of five such markers: (i) the EDA form of fibronectin (Fn(EDA)), (ii) lysyl oxidase (LOX), (iii) lysyl oxidase-like protein 2 (LoxL2), (iv) connective tissue growth factor (CTGF, CCNII), and (v) the N-terminal propeptide of type III procollagen (PIIINP).

Use of fructosyl peptide oxidase for HbA1c assay.

ARKRAY, Inc developed the world's first automatic glycohemoglobin analyzer based on HPLC (1981). After that, ARKRAY developed enzymatic HbA1c assay "CinQ HbA1c" with the spread and diversification of HbA1c measurement (2007). CinQ HbA1c is the kit of Clinical Chemistry Analyzer, which uses fructosyl peptide oxidase (FPOX) for a measurement reaction. This report mainly indicates the developmental background, measurement principle, and future of the enzymatic method HbA1c reagent.

Serum lysyl oxidase-like 2 levels and idiopathic pulmonary fibrosis disease progression.

We evaluated whether lysyl oxidase-like 2 (LOXL2), which promotes cross-linking of collagen in pathological stroma, was detectable in serum from idiopathic pulmonary fibrosis (IPF) patients, and assessed its relationship with IPF disease progression. Patients from the ARTEMIS-IPF (n=69) and the Genomic and Proteomic Analysis of Disease Progression in IPF (GAP) (n=104) studies were analysed. Baseline serum LOXL2 (sLOXL2) levels were compared with baseline clinical and physiological surrogates of disease severity, and the association with IPF disease progression was assessed using a classification and regression tree (CART) method. sLOXL2 correlated weakly with forced vital capacity and carbon monoxide diffusion capacity (r -0.24-0.05) in both cohorts. CART-determined thresholds were similar: ARTEMIS-IPF 800 pg·mL(-1) and GAP 700 pg·mL(-1). In ARTEMIS-IPF, higher sLOXL2 (>800 pg·mL(-1)) was associated with increased risk for disease progression (hazard ratio (HR) 5.41, 95% CI 1.65-17.73). Among GAP subjects with baseline spirometric data (n=70), higher sLOXL2 levels (>700 pg·mL(-1)) were associated with more disease progression events (HR 1.78, 95% CI 1.01-3.11). Among all GAP subjects, higher sLOXL2 levels were associated with increased risk for mortality (HR 2.28, 95% CI 1.18-4.38). These results suggest that higher sLOXL2 levels are associated with increased risk for IPF disease progression. However, due to multiple limitations, these results require validation.

Validation of four candidate pancreatic cancer serological biomarkers that improve the performance of CA19.9.

BACKGROUND: The identification of new serum biomarkers with high sensitivity and specificity is an important priority in pancreatic cancer research. Through an extensive proteomics analysis of pancreatic cancer cell lines and pancreatic juice, we previously generated a list of candidate pancreatic cancer biomarkers. The present study details further validation of four of our previously identified candidates: regenerating islet-derived 1 beta (REG1B), syncollin (SYCN), anterior gradient homolog 2 protein (AGR2), and lysyl oxidase-like 2 (LOXL2). METHODS: The candidate biomarkers were validated using enzyme-linked immunosorbent assays in two sample sets of serum/plasma comprising a total of 432 samples (Sample Set A: pancreatic ductal adenocarcinoma (PDAC, n = 100), healthy (n = 92); Sample Set B: PDAC (n = 82), benign (n = 41), disease-free (n = 47), other cancers (n = 70)). Biomarker performance in distinguishing PDAC from each control group was assessed individually in the two sample sets. Subsequently, multiparametric modeling was applied to assess the ability of all possible two and three marker panels in distinguishing PDAC from disease-free controls. The models were generated using sample set B, and then validated in Sample Set A. RESULTS: Individually, all markers were significantly elevated in PDAC compared to healthy controls in at least one sample set (p ≤ 0.01). SYCN, REG1B and AGR2 were also significantly elevated in PDAC compared to benign controls (p ≤ 0.01), and AGR2 was significantly elevated in PDAC compared to other cancers (p < 0.01). CA19.9 was also assessed. Individually, CA19.9 showed the greatest area under the curve (AUC) in receiver operating characteristic (ROC) analysis when compared to the tested candidates; however when analyzed in combination, three panels (CA19.9 + REG1B (AUC of 0.88), CA19.9 + SYCN + REG1B (AUC of 0.87) and CA19.9 + AGR2 + REG1B (AUC of 0.87)) showed an AUC that was significantly greater (p < 0.05) than that of CA19.9 alone (AUC of 0.82). In a comparison of early-stage (Stage I-II) PDAC to disease free controls, the combination of SYCN + REG1B + CA19.9 showed the greatest AUC in both sample sets, (AUC of 0.87 and 0.92 in Sets A and B, respectively). CONCLUSIONS: Additional serum biomarkers, particularly SYCN and REG1B, when combined with CA19.9, show promise as improved diagnostic indicators of pancreatic cancer, which therefore warrants further validation.

Avoidance to wipe alcohol before venipuncture is not a source of spurious hemolysis.

BACKGROUND: It is still uncertain whether or not avoidance to let disinfectant alcohol dry at the site of venipuncture is a source of spurious hemolysis when drawing venous blood. METHODS: In a consecutive series of 52 outpatients referred for routine laboratory testing, venous blood was drawn by direct venipuncture with (odd group) or without (pair group) wiping 70% isopropyl alcohol at the site of venipuncture. A 3.5 mL evacuated tube with dot activator and gel separator was drawn from a vein of the upper limb, serum was immediately separated with standard centrifugation and tested for potassium, lactate dehydrogenase (LD), aspartate aminotransferase (AST) and hemolysis index (HI) on Roche Cobas. RESULTS: No specimen was discarded for unsatisfactory venipuncture. No differences for age and gender were observed between groups. As regards the four parameters investigated, no significant differences could be observed between patients in whom blood was drawn with or without letting the alcohol dry. It is also noteworthy that no sample in both groups exceeded the conventional sample rejection threshold of cell-free hemoglobin. CONCLUSIONS: The results of our prospective, randomized study attest that failure to wipe alcohol at the site of venipuncture should not be considered as a potential source of spurious hemolysis when drawing blood.

The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.

Decreased total antioxidants status in the plasma of patients with pseudoexfoliation glaucoma.

PURPOSE: To evaluate total antioxidant status (TAS) in the plasma of pseudoexfoliation glaucoma (PEG) patients and to compare this level with a matching control group. Additionally, we aim to investigate the effect of the combined action of the lysyl oxidase-like 1 (LOXL1) mutation status with TAS level on the development of PEG. METHODS: Plasma samples were obtained from 54 PEG patients and 54 controls of matching age, sex, and ethnicity. TAS in all samples was determined by spectrophotometric and enzyme-linked immunosorbent assay methods. The coding region of LOXL1, where it encompasses both single nucleotide polymorphisms (SNPs; rs1048661 and rs3825942), was sequenced. RESULTS: The mean (±SD) total antioxidant (TAS) value was lower among patients: 0.87 (0.24), range 0.9-1.41 than controls: 1.07 (0.23), range 0.72-1.94, and this difference was statistically significant (p<0.0001: 95%CI: -0.295-0.114). Evaluating the impact of age, sex, and the mutation in addition to the mean TAS value in patients with PEG, a logistic regression analysis was conducted using diseased/not diseased as the outcome of interest (the dependent variable). Results show that, controlling for all other variables, mean TAS value (p<0.0001) and the mutation G/G in rs3825942 (p=0.041) are significant risk factors for PEG. CONCLUSIONS: Our findings provide evidence that TAS decreases in the plasma of PEG patients, suggesting that TAS may have an important role in the pathogenesis of PEG. The combined effect of the "G" allele and the decreased TAS may contribute to the overall pathogenesis of PEG.

Clinical evaluation of serum alcohol dehydrogenase activity in horses with acute intestinal obstruction.

OBJECTIVES: To measure serum alcohol dehydrogenase (ADH) activity in horses with acute intestinal obstruction and to determine the diagnostic and prognostic utility of this analyte. DESIGN: Prospective observational study. SETTING: University Veterinary Hospital. ANIMALS: Thirty healthy horses (control group) and 77 horses with acute intestinal obstruction, including 36 horses with nonstrangulating obstruction (23 with left ventral colon impaction and 13 with left dorsal displacement [G1], 22 with small intestinal strangulation [G2], and 19 with colon torsion [G3]). INTERVENTIONS: Serum ADH activity was assayed spectrophotometerically in all horses. Serum lactate concentration and hepatic enzyme (aspartate aminotransferase, gamma-glutamyl transferase, glutamate dehydrogenase) activities were measured using an automatic analyzer. MEASUREMENTS AND MAIN RESULTS: The median [interquartile range] serum ADH activity in healthy horses was 10.5 [8.7-11 U/L]. ADH activity was significantly increased (P<0.05) in G1=16.5 [13.8-18 U/L], G2=40 [20-74.9 U/L], and G3=63.2 [40-78 U/L] compared with healthy controls. Aspartate aminotransferase and glutamate dehydrogenase activities were also significantly increased in G3 in comparison with controls. ADH activity was correlated with serum lactate concentration in G1 and G3, respectively (P<0.01, r=0.55 and 0.8). Other liver enzymes did not show any significant correlation with lactate. ADH activity was directly related to the probability of strangulation; odds ratio=1.11. ADH activity >20 U/L had 80.6% specificity and 80.5% sensitivity for discriminating horses with strangulating obstruction. Twelve horses euthanized before surgery were excluded from the outcome analysis. Increasing ADH activity was associated with nonsurvival; odds ratio=1.03. ADH activity <80 U/L had 94.44% specificity and 66.67% sensitivity for survival. CONCLUSION: Serum ADH activity may be a useful clinical parameter in detecting intestinal strangulation in horses and may provide some prognostic value in horses with acute intestinal obstruction.

Activities of enzymes in the malate-aspartate shuttle and the isoenzyme pattern of lactate dehydrogenase in plasma and peripheral leukocytes of lactating Holstein cows and riding horses.

The activities of the enzymes involved in the malate-aspartate shuttle and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in plasma and peripheral leukocytes of lactating Holstein cows and thoroughbred riding horses as representative herbivorous animals. In the horse plasma, LDH activities were significantly lower and AST activities were significantly higher than those in the cow plasma. The specific activities of cytosolic malate dehydrogenase (MDH), LDH and AST in the horse leukocytes were higher than those in the cows. The cytosolic ratio of MDH/LDH activity (ML ratio) in the horse leukocytes was significantly lower than that in the cow leukocytes owing to significantly higher activities of LDH. The ML ratio was considered to reflect the difference in energy metabolism in leukocytes between cows and horses. The plasma LDH isoenzyme patterns of cow and horse showed the characteristic as herbivorous animals with dominance of LDH-1, -2 and -3. The LDH isoenzyme patterns with dominance of LDH-3 and -4 in the horse leukocytes were remarkably different from those in the cow leukocytes. There were significant differences in activities of malate-aspartate shuttle enzymes, ML ratio and LDH isoenzyme patterns in the cytosolic fractions of leukocytes between the lactating cows and the riding horses.