Blood-based gene expression profile of oxidative stress and antioxidant genes for identifying surrogate markers of liver tissue injury in chronic hepatitis C patients.

Oxidative stress is the process by which reactive molecules and free radicals are formed in cells. In this study, we report the blood-based gene expression profile of oxidative stress and antioxidant genes for identifying surrogate markers of liver tissue in chronic hepatitis C (CHC) patients by using real-time PCR. A total of 144 untreated patients diagnosed with CHC having genotype 3a and 20 healthy controls were selected for the present study. Liver biopsy staging and grading of CHC patients were performed using the METAVIR score. Total RNA was extracted from liver tissue and blood samples, followed by cDNA synthesis and real-time PCR. The relative expression of genes was calculated using the ΔΔCt method. The expression profile of 84 genes associated with oxidative stress and antioxidants was determined in liver tissue and blood samples. In liver tissue, 46 differentially expressed genes (upregulated, 27; downregulated, 19) were identified in CHC patients compared to normal samples. In blood, 61 genes (upregulated, 51; downregulated; 10) were significantly expressed in CHC patients. A comparison of gene expression in liver and whole blood showed that 20 genes were expressed in a similar manner in the liver and blood. The expression levels of commonly expressed liver and blood-based genes were also correlated with clinical factors in CHC patients. A receiver operating curve (ROC) analysis of oxidative stress genes (ALB, CAT, DHCR24, GPX7, PRDX5, and MBL2) showed that infections in patients with CHC can be distinguished from healthy controls. In conclusion, blood-based gene expression can reflect the behavior of oxidative stress genes in liver tissue, and this blood-based gene expression study in CHC patients explores new blood-based non-invasive biomarkers that represent liver damage.

Study on the effect of black cumin (Nigella sativa Linn.) on experimental renal ischemia-reperfusion injury in rats

PURPOSE: To evaluate the effect of Black cumin (Nigella sativa Linn.) pre-treatment on renal ischemia/reperfusion (I/R) induced injury in the rats.METHODS: A total of 40 male Wistar rats were randomly allocated into five equal groups including Sham, I/R model and three I/R+ Black cumin (0.5, 1 and 2%)-treated groups. I/R groups' kidneys were subjected to 60 min of global ischemia at 37°C followed by 24 h of reperfusion. At the end of reperfusion period, the rats were euthanized. Superoxide dismutase, catalase and glutathione peroxidase activities as well as reduced glutathione and renal malondialdehyde contents were determined in renal tissues. Kidney function tests and histopathological examination were also performed.RESULTS: High serum creatinine, blood urea nitrogen and uric acid as well as malondialhehyde (MDA) levels, and low antioxidant enzyme activities were observed in I/R rats compared to the sham rats. Pre-treatment with Black cumin for three weeks prior to IR operation improved renal function and reduced I/R induced renal inflammation and oxidative injury. These biochemical observations were supported by histopathological test of kidney sections.CONCLUSION:Black cumin significantly prevented renal ischemia/reperfusion induced functional and histological injuries.

Clinical evaluation with self-sequential longitudinal reference intervals: pregnancy outcome and neonatal thyroid stimulating hormone level associated with maternal thyroid diseases / Evaluación clínica con intervalos de referencia longitudinal auto-secuencial: evolución clínica del embarazo y niveles de la hormona estimulante de la tiroides en neonatos asociados con enfermedades tiroideas maternas

OBJECTIVE: We attempted to evaluate maternal thyroid function in a new self-sequential longitudinal reference interval (SLRI) which we established recently. By this method, we analysed the correlation between pregnancy outcome, neonatal thyroid stimulating hormone (TSH) level and maternal thyroid diseases. METHODS: A total of 1744 pregnant women participated in the study and 1747 babies were born from those women (three bore twins). The levels of TSH, free thyroxine (FT4) and thyroid peroxidase antibodies (TPO-Ab) of mothers were quantified by electrochemistry immunoassay (ECL). The levels of neonatal blood TSH were detected by time-resolved fluorescence immunoassay (TRFIA). All data were collected and statistically analysed by SPSS 13.0 software. RESULTS: With our new SLRI method, we found that 0.11%~3.84% pregnant women would get thyroid diseases. Subclinical hypothyroidism was the most common maternal thyroid disorder. Being positive for thyroid peroxidase antibodies was a significant risk factor of subclinical hypothyroidism during pregnancy. The median, P2.5~P97.5, and interquartile range (IQR) of neonatal TSH (N-TSH) of 1747 babies were 2.72 mIU/L, 0.10~8.01 mIU/L and 2.62 mIU/L, respectively; 28.6% of pregnant women with thyroid diseases developed pregnancy complications. The prevalence was significantly higher than in the normal thyroid function group (p< 0.001). The levels of N-TSH were low correlated with maternal TSH levels (p < 0.05), but there were no significant correlations between N-TSH and maternal FT4 and maternal TPO-Ab (p > 0.05). CONCLUSIONS: Thyroid disorders, especially subclinical hypothyroidism, are common in pregnant women. These disorders are associated with pregnancy and fetal outcome. Routine maternal thyroid function screening is important and should be recommended.

OBJETIVO: Intentamos evaluar la función tiroidea materna en un nuevo intervalo de referencia longitudinal auto-secuencial (SLRI) que establecimos recientemente. Por este método, analizamos la correlación entre el resultado del embarazo, el nivel de la hormona estimulante de la tiroides (TSH) en neonatos, y las enfermedades tiroideas maternas MÉTODOS: Un total de 1744 mujeres embarazadas participó en el estudio y 1747 bebés nacieron de esas mujeres (tres de ellas tuvieron gemelos). Los niveles de TSH, la tiroxina libre (FT4), y los anticuerpos de la peroxidasa tiroidea (TPO-Ab) de las madres, fueron cuantificados mediante inmunoensayo electroquímico (ECL). Los niveles de TSH en la sangre de los neonatos, fueron determinados mediante inmunoensayo por fluorescencia resuelto en el tiempo (TRFIA). Todos los datos fueron recogidos y analizados estadísticamente usando el software SPSS 13.0 RESULTADOS: Con nuestro nuevo método SLRI, encontramos que 0.11%~3.84% de las mujeres embarazadas contraerán enfermedades tiroideas. El hipotiroidismo subclínico fue el trastorno de la tiroides materna más común. Ser positivo a los anticuerpos de la peroxidasa tiroidea fue un factor de riesgo significativo del hipotiroidismo subclínico durante el embarazo. La mediana, P2.5~P97.5, y el rango intercuartil (IQR) de la TSH (N-TSH) neonatal de los 1747 bebés fueron 2.72 mIU/L, 0.10~8.01 mIU/L y 2.62 mIU/L respectivamente. El 28.6% de las mujeres embarazadas que tenían enfermedades tiroideas, desarrollaron complicaciones del embarazo. La prevalencia fue significativamente más alta que en el grupo con función tiroidea normal (p < 0.001). Los niveles de N-TSH fueron bajos en correlación con los niveles de TSH maternos (p < 0.05), pero no hubo ninguna correlación significativa entre la N-TSH y la FT4 materna, y la TPO-Ab materna (p > 0.05). CONCLUSIÓNS: Los trastornos tiroideos, especialmente el hipotiroidismo, son comunes en las mujeres embarazadas.Estos trastornos se hallan asociados con el resultado del embarazo y el resultado fetal. El tamizaje de rutina de la función tiroidea materna es importante y debe recomendarse.

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions.

Members of the peroxidase-cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase-cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase-cyclooxygenase family, including the catalysis of dityrosine formation.

Relevance of non-ceruloplasmin copper to oxidative stress in patients with hepatocellular carcinoma.

Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent pro-oxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n = 18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n = 20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper.

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2.

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K(M) of 2.4 microM for human thioredoxin and a very low K(M) for H2O2 (0.7 microM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H2O2 or peroxynitrite, were determined by competition kinetics, k(2) = 1.0 x 10(8) and 1.4 x 10(7) M(-1) s(-1) at 25 degrees C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K(d) < 10(-23) M(4) which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.

Expression of peroxiredoxin I in plasma cells of oral inflammatory diseases.

The participation of reactive oxygen species (ROS) in the immune response, both as pathogen killers and as mediators of signaling pathways, is well established. However, little is known about the enzymes responsible for ROS elimination in immune cells. Peroxiredoxin I (PrdxI) is a multifunctional enzyme that exhibits thioredoxin-dependent peroxidase activity. It has been described as a major hydrogen peroxide (H(2)O(2))-inducible protein in mouse peritoneal macrophages. In order to characterize its participation in the antioxidant defense of inflammatory/immune cells in greater detail, we evaluated its expression at sites of the oral cavity affected by inflammatory disorders induced by different agents (infectious, chemical, mechanical or tumor). In this study we demonstrated, by immunohistochemistry, that PrdxI is expressed in plasma cells, but not in B lymphocytes, regardless of the inflammation-inducing agent. We suggest that PrdxI induction could be considered a crucial part of the cellular adaptive response to the B-cell differentiation process to cope with the additional H(2)O(2) associated with massive disulfide bond formation during immunoglobulin folding in the endoplasmic reticulum of plasma cells. PrdxI could diminish the tissue damage that accompanies inflammation.

Proteomics-based approach identifying autoantibody against peroxiredoxin VI as a novel serum marker in esophageal squamous cell carcinoma.

PURPOSE: Detection of novel tumor-related antigens and autoantibodies will aid in diagnosis of early-stage cancer and in development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens in an esophageal squamous cell carcinoma (ESCC) cell line (TE-2) and related autoantibodies in sera from patients with ESCC using a proteomics-based approach. EXPERIMENTAL DESIGN: TE-2 proteins were separated by two-dimensional PAGE, followed by Western blot analysis in which sera of patients with ESCC, healthy controls, and patients with other cancers were tested for primary antibodies. Positive spots were excised from silver-stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). RESULTS: Sera from patients with ESCC yielded multiple spots, one of which was identified as peroxiredoxin (Prx) VI by MALDI-TOF/TOF MS. Western blot analysis against recombinant Prx VI showed reactivity in sera from 15 of 30 (50%) patients with ESCC and 2 of 30 (6.6%) healthy individuals. Autoantibody against Prx VI was found in sera from 1 of 30 (3.3%) patients with other types of cancer (colon cancer). CONCLUSION: We have identified for the first time an autoantibody against Prx VI in ESCC patients. The proteomic approach implemented here offers a powerful tool for identifying novel serum markers that may display clinical usefulness against cancer.

Peripheral markers of oxidative stress in chronic mercuric chloride intoxication

The present study was designed to evaluate the time course changes in peripheral markers of oxidative stress in a chronic HgCl2 intoxication model. Twenty male adult Wistar rats were treated subcutaneously daily for 30 days and divided into two groups of 10 animals each: Hg, which received HgCl2 (0.16 mg kg-1 day-1), and control, receiving the same volume of saline solution. Blood was collected at the first, second and fourth weeks of Hg administration to evaluate lipid peroxidation (LPO), total radical trapping antioxidant potential (TRAP), and superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and catalase (CAT). HgCl2 administration induced a rise (by 26 percent) in LPO compared to control (143 ± 10 cps/mg hemoglobin) in the second week and no difference was found at the end of the treatment. At that time, GST and GPx were higher (14 and 24 percent, respectively) in the Hg group, and Cu,Zn-SOD was lower (54 percent) compared to control. At the end of the treatment, Cu,Zn-SOD and CAT were higher (43 and 10 percent, respectively) in the Hg group compared to control (4.6 ± 0.3 U/mg protein; 37 ± 0.9 pmol/mg protein, respectively). TRAP was lower (69 percent) in the first week compared to control (43.8 ± 1.9 mM Trolox). These data provide evidence that HgCl2 administration is accompanied by systemic oxidative damage in the initial phase of the process, which leads to adaptive changes in the antioxidant reserve, thus decreasing the oxidative injury at the end of 30 days of HgCl2 administration. These results suggest that a preventive treatment with antioxidants would help to avoid oxidative damage in subjects with chronic intoxication.

ALDH2 and CYP2E1 genotypes, urinary acetaldehyde excretion and the health consequences in moderate alcohol consumers.

Deficiencies in mitochondrial low-Km aldehyde dehydrogenase (ALDH2) activity, and consequently high blood acetaldehyde levels, have been suggested to relate to various diseases in Japanese, including esophageal cancer. In the present study, 200 men aged 35-59 years randomly selected from an occupational population were analyzed for the association of ALDH2 genotypes and cytochrome P450-2E1 (CYP2E1) genotypes with the urinary excretion of acetaldehyde (which is bound to some chemicals in the urine) and with common alcohol-related health consequences. Urinary acetaldehyde excretion was increased, reflecting increased alcohol consumption even in this moderate alcohol-consuming population. Neither the ALDH2 nor the CYP2E1 genotypes showed significant influence on the elevation of urinary acetaldehyde excretion. Neither these genotypes nor urinary acetaldehyde concentration significantly affected blood pressure, serum aspartate aminotransferase and gamma-glutamyl transferase activities, or serum HDL-cholesterol and lipid peroxide concentrations. It was concluded that acetaldehyde accumulates in moderate alcohol consumers irrespective of ALDH2 and CYP2E1 genotype, and that the implications of these genotypes and acetaldehyde accumulation in terms of common alcohol-related health consequences were obscure. The results also suggest that the carcinogenicity of acetaldehyde on esophageal mucosa depends greatly upon repeated exposure to high blood acetaldehyde, even through transient rather than chronic exposure.

Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection.

Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.

Changes in inflammatory markers following treatment of acute exacerbations of obstructive pulmonary disease.

The aim ofthe study was to investigate changes in inflammatory markers following emergency treatment of obstructive pulmonary disease. The study comprised 43 patients. After acute treatment, they were given either 30 mg of prednisolone p.o. or 1600 microg of inhaled budeson de daily for 1 week. Over the following 3 weeks, all the patients were given 1600 microg of inhaled budesonide daily. Blood samples for measurements of eosinophil cationic protein (S-ECP), eosinophil peroxidase (S-EPO), total eos nophil count (B-Eos), myeloperoxidase (S-MPO) and human neutrophil lipocaline (HNL) were taken and spirometry was performed before emergency treatment and after 1 and 4 weeks. There was no difference in the improvement in forced expiratory volume in 1 sec (FEV1) between patients given prednisolone or budesonide. Patients with an improvement in FEV1 of >20% of baseline after 1 and 4 weeks displayed a larger decrease in eosinophil markers. The correlation between deltaFEV1 and deltaS-ECP was r= -0.37, P < 0.05, deltaS-EPO -0.40, P < 0.01 and deltaB-Eos -0.44, P < 0.01, after 4 weeks. This correlation was highly significant in patients who had smoked < or = 5 pack-years, while the correlation was not significant in patients with a longer smoking history and chronic airflow limitation (best FEV <80% of predicted). We conclude that the change in eosinophil markers is correlated to the improvement in lung function in non-smokers or short-term smokers following the emergency treatment of obstructive pulmonary disease. This study indicates that following eosinophil markers is more useful in patients with asthma than patients with COPD.

Eosinophil markers in blood, serum, and urine for monitoring the clinical course in childhood asthma: impact of budesonide treatment and withdrawal.

BACKGROUND: Markers of airway inflammation are needed for prediction of asthma deterioration and evaluation of disease severity. Few studies have focused on the dynamics of airway inflammation as reflected by the activity of the eosinophils and their proteins after withdrawal of inhaled corticosteroids. OBJECTIVE: Our goal was to investigate the effect of withdrawal of inhaled budesonide on eosinophil count in blood and eosinophil proteins in serum and urine and to relate the levels of these markers to the risk of symptoms of asthma, increased bronchial hyperresponsiveness, and deterioration of lung function. METHODS: Thirty-three children were randomly selected to continue or discontinue use of inhaled budesonide in a double-blind, placebo-controlled study. They were followed up for 4 months with regular analysis of blood, serum, and urine samples; lung function; and methacholine challenges. Eosinophil activity markers were analyzed. Age-matched healthy children provided reference data for all parameters measured. RESULTS: The eosinophil number in blood and eosinophil protein levels in serum (serum eosinophil cationic protein [ECP] and serum eosinophil peroxidase [EPO]) increased significantly in the withdrawal group, and the difference between the groups was significant (P =.02 for all). Twenty-nine percent of the children in the withdrawal group remained symptom free. This subgroup had eosinophil counts at baseline below 350/microL, a serum ECP level below 15 microg/L, and a serum EPO level below 25 microg/L, each of which was related to a low risk of exacerbation (relative risk = 0.37, 0.48, and 0.37 respectively; P <.05 for all). All eosinophil markers were lower in the healthy children than in the symptom-free children with asthma. CONCLUSION: Our data indicate that eosinophil count and/or ECP and EPO levels can be used to estimate the short-term risk of deterioration and the need for corticosteroid treatment in cases of mild and moderate allergic asthma.

Neutrophil and eosinophil granule proteins as markers of response to local prednisolone treatment in distal ulcerative colitis and proctitis.

OBJECTIVE: The pathophysiological role of neutrophil and eosinophil granulocytes in relation to steroid enema treatment was studied in patients with distal ulcerative colitis and proctitis. METHODS: The rectal release of the neutrophil (myeloperoxidase, MPO), and eosinophil (eosinophilic cationic protein, ECP and eosinophil peroxidase, EPO) granule constituents were measured in 11 patients using intraluminal segmental perfusion of the rectum. The released amounts of MPO, ECP, and EPO in the perfusion fluids were determined by radioimmunoassays before and during prednisolone enema treatment and related to clinical, endoscopical, and histopathological data in addition to treatment outcome. RESULTS: Clinical activity and particularly endoscopic activity correlated well with intraluminal MPO concentrations both before and during treatment. At the end of the study, eight of 11 patients fulfilled predefined response criteria; all responding patients had significant decrease of MPO concentrations (p < 0.01). This decline of MPO concentration was seen after 7 days of treatment (p < 0.05) in the response group and often occurred before clinical improvement. There was a nonsignificant trend toward a decrease in the concentrations of ECP and EPO at the end of treatment in responders.

Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.

BACKGROUND: The peroxiredoxins (Prxs) are an emerging family of multifunctional enzymes that exhibit peroxidase activity in vitro, and in vivo participate in a range of cellular processes known to be sensitive to reactive oxygen species. Thioredoxin peroxidase B (TPx-B), a 2-Cys type II Prx from erythrocytes, promotes potassium efflux and down-regulates apoptosis and the recruitment of monocytes by endothelial tissue. RESULTS: The crystal structure of human decameric TPx-B purified from erythrocytes has been determined to 1.7 [corrected)] A resolution. The structure is a toroid comprising five dimers linked end-on through predominantly hydrophobic interactions, and is proposed to represent an intermediate in the in vivo reaction cycle. In the crystal structure, Cys51, the site of peroxide reduction, is oxidised to cysteine sulphinic acid. The residue Cys172, lies approximately 10 A away from Cys51 [corrected]. CONCLUSIONS: The oxidation of Cys51 appears to have trapped the structure into a stable decamer, as confirmed by sedimentation analysis. A comparison with two previously reported dimeric Prx structures reveals that the catalytic cycle of 2-Cys Prx requires significant conformational changes that include the unwinding of the active-site helix and the movement of four loops. It is proposed that the stable decamer forms in vivo under conditions of oxidative stress. Similar decameric structures of TPx-B have been observed by electron microscopy, which show the protein associated with the erythrocyte membrane.

The induction of cyclooxygenase-2 (COX-2) in cultured endothelial cells treated with serum from preeclampsia is mediated by interleukin-6.

COX-2 protein, but not COX-1 protein, was induced in HUVEC from women with a normal pregnancy (nHUVEC) treated with serum from patients with preeclampsia (pSerum), but not with serum from women with a normal pregnancy (nSerum). COX activity in pSerum treated nHUVEC was less than in nSerum treated nHUVEC. Interestingly, the induction of COX-2 protein in nHUVEC treated with pSerum was inhibited by antiIL-6 antibody. The decreased COX activity in nHUVEC treated with pSerum plus antiIL-6 antibody was also reversed in a dose dependent manner. Thus, the induction of COX-2 in pSerum treated nHUVEC was mediated by IL-6. Therefore, the development of selective inhibitors of COX-2 or of IL-6 antagonists may have a potential role in the prevention and treatment of preeclampsia.

Crystallization and preliminary X-ray analysis of human thioredoxin peroxidase-B from red blood cells.

Two different crystal forms of human thioredoxin peroxidase-B have been grown by vapour diffusion using polyethylene glycol 400 as a precipitant. Monoclinic P21 crystals were grown from freshly purified protein, whilst orthorhombic P212121 crystals were grown from purified protein that had been stored in ammonium sulfate, but otherwise under the same conditions. The diffraction from both crystal forms was observed to extend to beyond 2.0 A resolution using synchrotron radiation. Complete native data sets to 1.8 and 3. 7 A have been collected from the monoclinic and orthorhombic crystals, respectively.

Antioxidant enzymes, inflammatory indices and lifestyle factors in older men: a cohort analysis.

We examined the relationship between blood antioxidant enzyme activities, indices of inflammatory status and a number of lifestyle factors in the Caerphilly prospective cohort study of ischaemic heart disease. The study began in 1979 and is based on a representative male population sample. Initially 2512 men were seen in phase I, and followed-up every 5 years in phases II and III; they have recently been seen in phase IV. Data on social class, smoking habit, alcohol consumption were obtained by questionnaire, and body mass index was measured. Antioxidant enzyme activities and indices of inflammatory status were estimated by standard techniques. Significant associations were observed for: age with alpha-1-antichymotrypsin (p < 0.0001) and with caeruloplasmin, both protein and oxidase (p < 0.0001); smoking habit with alpha-1-antichymotrypsin (p < 0.0001), with caeruloplasmin, both protein and oxidase (p < 0.0001) and with glutathione peroxidose (GPX) (p < 0.0001); social class with alpha-1-antichymotrypsin (p < 0.0001), with caeruloplasmin both protein (p < 0.001) and oxidase (p < 0.01) and with GPX (p < 0.0001); body mass index with alpha-1-antichymotrypsin (p < 0.0001) and with caeruloplasmin protein (p < 0.001). There was no significant association between alcohol consumption and any of the blood enzymes measured. Factor analysis produced a three-factor model (explaining 65.9% of the variation in the data set) which appeared to indicate close inter-relationships among antioxidants.

Nerve growth factor is preformed in and activates human peripheral blood eosinophils.

BACKGROUND: Recent studies have shown that nerve growth factor (NGF) is produced by and can act on several immune-inflammatory cells. OBJECTIVES: The objective of this study was to study the effects of NGF on human peripheral blood eosinophils and assess whether these cells produce and store NGF. METHODS: Eosinophils were purified by negative immunoselection (magnetic cell sorting systems, purity 98% to 100%) from 13 subjects (9 to 26 years old) with mild blood eosinophilia, mainly of allergic origin. Eosinophils were incubated with NGF (50 to 1000 ng/mL), and supernatants were collected for measurement of eosinophil peroxidase (EPO, 20 minutes, colorimetric enzymatic assay) and IL-6 (12 hours, ELISA). Eosinophil viability was evaluated by Trypan blue test (days 2, 3, and 4). NGF content in freshly isolated eosinophils, after ultrasound disruption, was determined with a 2-site immunoenzymatic assay. The presence of mRNA for NGF was evaluated by reverse transcription PCR. RESULTS: NGF caused EPO release (highly significant at 1000 ng/mL NGF). IL-6 release from eosinophils was not higher than IL-6 spontaneously released into culture medium alone. NGF did not significantly affect the number of viable eosinophils. NGF was found in the eosinophil sonicates (1.5 to 17.8 pg/mL per 106 cells). Similarly, mRNA for NGF was detected by reverse transcription PCR in the freshly isolated eosinophils. CONCLUSIONS: NGF activates human peripheral blood eosinophils from subjects with mild eosinophilia to selectively release inflammatory mediators. Eosinophils store and produce NGF. Therefore the capability of NGF to induce a secretory response and its production and storage by circulating human eosinophils suggest a possible role for NGF in conditions associated with eosinophilia, including allergic disease.