Stress In Leukocytes Research Articles

Indicators of oxidative stress in leukocytes isolated from bottlenose dolphins (Tursiops truncatus) in response to a proinflammatory challenge

Few studies have analyzed the indicators of oxidative stress in marine mammals following exposure to lipopolysaccharides (LPS); sex and maturity-related differences have not been explored. The objective of this study was to compare the indicators of oxidative stress following exposure to LPS for 24 and 48 h in isolated Pacific bottlenose dolphin (Tursiops truncatus; N = 12) leukocytes in relation to sex and maturity stage, using spectrophotometry. Following 48 h under experimental conditions (10 μg LPS mL−1), the leukocytes from males (n = 5) produced significantly more superoxide radical (O2•-; F (1, 8) = 13.965, p = 0.006) and displayed significantly greater activities of catalase (CAT; F (1, 8) = 9.465, p = 0.015) and glutathione S-transferase (GST; p = 0.028) compared to the leukocytes from females (n = 7). Following 48 h under experimental conditions, maturity-stage did not significantly influence the indicators of oxidative stress. Mature bottlenose dolphins (n = 7) had a significantly higher average daily dietary intake compared to immature bottlenose dolphins (n = 5; F (1, 10) = 5.825, p = 0.036). These results suggest that sex-related strategies for coping with a proinflammatory challenge may be present within the leukocytes from bottlenose dolphins, while potential maturity stage-related strategies require further investigation.

Endoplasmic reticulum stress in leukocytes from phenylketonuric patients

Abstract Objectives Phenylketonuria (PKU) is a proteinopathy due to the deficiency of phenylalanine hydroxylase (PAH) enzyme. The pathological elevation of phenylalanine (Phe) and its metabolites in PKU is linked to neurological hallmarks and mental disabilities. The aim of this study was to examine the hypothesis that high levels of Phe caused endoplasmic reticulum (ER) stress in PKU patients. Methods We primarily evaluated ER stress markers glucose-regulated protein78 (GRP78) and C/-EBP homologous protein (CHOP), and thiobarbituric acid-reactive substances (TBARS) as a biomarker of oxidative stress in leukocytes and correlated it with blood Phe values from patients with PKU. Patients in this study were selected from individuals who were diagnosed with PKU as a result of the national neonatal screening program and undergone treatment at our university hospital. The subjects were divided into four groups: healthy controls, patients with hyperphenylalaninemia (HPA), BH4-responsive patients with PKU and patients with classic PKU. GRP78, CHOP and TBARS levels were estimated in leukocytes isolated from whole blood of subjects, Phe and tyrosine levels were determined in plasma. Results The levels of Phe in BH4-responsive PKU and classic PKU groups were statistically higher as compared to healthy controls, and Phe levels were higher in classic PKU compared to HPA group. CHOP levels were elevated by 35.3% in BH4-responsive group compared to control. GRP78, CHOP and TBARS showed no statistical differences between control and patient groups. GRP78 was also negatively correlated with Phe levels. Conclusions These results suggested that blood Phe concentrations might not be associated to ER stress in white blood cells obtained from the PKU patient groups under treatment.

EVALUATION OF THE LEUKOCYTE INDEX IN SICKLE CELL ANEMIA WITH THE USE OF HYDROXYUREA

The term sickle cell disease (SCD) is used to describe a group of genetic disorders in which there is a predominance of HbS, a variant hemoglobin originated from a single nucleotide polymorphism (SNP) responsible for the exchange of a glutamic acid by valine in position 6 of ß-globin chain. The most severe form of the disease is homozygous (HbSS). Thus, sickle cell disease patients may present different clinical manifestations and inflammatory processes. A pharmacotherapeutic used for the treatment is Hydroxyurea. The action of this drug includes increasing fetal hemoglobin levels and improving clinical severity and hematological parameters. This study aimed to investigate hematological stress in patients with sickle cell anemia treated (or not) with hydroxyurea, and classified regarding the risk of severity by the Calculator of severity of sickle cell disease. DNA samples from 200 patients with sickle cell anemia, in steady state of the disease, using and not using hydroxyurea, were submitted to molecular analysis to confirm HbSS. Then, chronic stress was characterized from the neutrophil-lymphocyte ratio (NLR) through the analysis of the blood count and clinical history. NLR is an important inflammatory marker, its calculation is indicated for patients with sickle cell disease and values above one may indicate hematological stress. Statistical analyzes were performed using IBM SPSS Statistics 20 software. Data normality was verified using the Shapiro-Wilk test. Assessed for non-normality, the non-parametric Wilcoxon test was performed to compare the means between the leukocyte profile and the use of hydroxyurea. Then, a comparison was made between the risk of death of patients who were (or not) treated with hydroxyurea. The results were shown as mean and standard deviation, with a significance level of 0.05. The means and standard deviation were NLR/With HU (1.76 ±1.53) and NLR/Without HU (1.65 ±1.07). In order to verify the difference between the means, we used the Wilcoxon test whose value was p = 0.975. Thus, we observed that there was no difference between leukocyte stress and the use of Hydroxyurea. When comparing the risk of death of patients which use the drug (1.96 ±0.73) and those who do not use it (2.22 ±0.75), we noticed that there were significant differences between the groups (p = 0.01). Therefore, the drug can contribute to reduce the risk of death. The non-relation between leukocyte stress and the use of hydroxyurea reported in this study is not well documented in the literature. However, we observed an increase in NLR in critically ill patients with sickle cell disease classified by the Calculator of severity of sickle cell disease. The role of hydroxyurea in the risk of life for patients with sickle cell anemia is well documented. It is known that it can help reducing the number of hospitalizations, length of stay, less occurrence of acute chest syndrome, lower morbidity and mortality rate, contributing to an increase of up to 40% in patient survival. Our results corroborate literature data. There was no relationship between stress through the NLR and the use of hydroxyurea, but there is a relationship between the severity of the disease and the use of the medication. Relating leukocyte stress with disease severity as well as the impact of hydroxyurea on these indices is important to understand the clinical manifestations of sickle cell anemia patients.

UV Light-Activated GdYVO4:Eu3+ Nanoparticles Induce Reactive Oxygen Species Generation in Leukocytes Without Affecting Erythrocytes In Vitro.

Nanoparticles (NPs) have been reported to be promising enhancement agents for radiation therapy. The aim of the study was to assess the cytotoxicity of UV non-treated and UV pretreated GdYVO4:Eu3+ nanoparticles against erythrocytes and leukocytes by detecting eryptosis and reactive oxygen species (ROS) generation. Levels of intracellular ROS in erythrocytes and leukocytes using a ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as eryptosis rate utilizing annexin V staining, following direct exposure to UV-activated and nonactivated NPs were detected by flow cytometry. Blood cells were collected from 9 intact WAG rats. Neither the UV light-untreated GdYVO4:Eu3+ NPs nor the treated ones promoted eryptosis and ROS generation in erythrocytes. Low concentrations of UV light-untreated NPs did not induce oxidative stress in leukocytes, evidenced by unaffected intracellular ROS levels. UV light treatment grants prooxidant properties to NPs, confirmed by NP-induced ROS overproduction in leukocytes. High concentrations of both UV light-treated and untreated NPs altered the redox state of leukocytes. UV light treatment imparts prooxidant properties to GdYVO4:Eu3+ NPs, making them promising radiosensitizing agents in cancer radiation therapy.

Brief High Oxygen Concentration Induces Oxidative Stress in Leukocytes and Platelets: A Randomized Cross-over Pilot Study in Healthy Male Volunteers.

Supplemental oxygen is administered routinely in the clinical setting to relieve or prevent tissue hypoxia, but excessive exposure may induce oxidative damage or disrupt essential homeostatic functions. It is speculated that oxidative stress in leukocytes and platelets may contribute to vascular diseases by promoting inflammation and cell aggregation. In this pilot study 30 healthy male volunteers (18-65 years) were exposed to high oxygen concentration (non-rebreather mask, 8 L/min, 100% O2) and synthetic air (non-rebreather mask, 8 L/min, 21% O2) in a cross-over design for 20 min at a 3-week interval. Venous blood samples were obtained at baseline and 1, 3, and 6 h postintervention. Primary outcome was generation of reactive oxygen species in leukocytes as measured by the redox-sensitive fluorescent dye dihydrorhodamine 123. Additional outcomes were oxidative stress in platelets and platelet aggregation as measured by thromboelastography (ROTEM) and Multiplate analyses. High oxygen exposure induced oxidative stress in leukocytes as evidenced by significantly higher mean fluorescence intensity (MFI) compared with synthetic air at 3 h postintervention (47% higher, P = 0.015) and 6 h postintervention (37% higher, P = 0.133). Oxidative stress was also detectable in platelets (33% higher MFI in comparison with synthetic air at 6 h, P = 0.024; MFI 20% above baseline at 3 h, P = 0.036; 37% above baseline at 6 h, P = 0.002). ROTEM analyses demonstrated reduced mean clotting time 1 h postintervention compared with baseline (-4%, P = 0.049), whereas there were no significant effects on other surrogate coagulation parameters. Clinically relevant oxygen exposure induces oxidative stress in leukocytes and platelets, which may influence the immune and clotting functions of these cells.

Loganic Acid, an Iridoid Glycoside Extracted from Cornus mas L. Fruits, Reduces of Carbonyl/Oxidative Stress Biomarkers in Plasma and Restores Antioxidant Balance in Leukocytes of Rats with Streptozotocin-Induced Diabetes Mellitus.

The various complications related to diabetes are due to the alteration in plasma components and functional activity of blood cells, hence the search for preventive remedies that would ameliorate the clinical condition of patients is a relevant problem today. The main aim of the present study was to examine the antidiabetic potency and antioxidant effects of loganic acid (LA) in blood of diabetic rats. LA showed a restoration of balance between functioning of antioxidant defense system and oxidative stress in leukocytes without notable effects on blood glucose levels when administered orally to rats (20 mg/kg b.w./day) for 14 days. LA ameliorated antioxidant status in leukocytes, as indicated by increasing the content of reduced glutathione and activities of catalase, glutathione peroxidase and glutathione reductase along with decreasing levels of intracellular reactive oxygen species. In addition, we observed the ability of LA to protect against formation and accumulation of glycation and oxidation protein products and malondialdehyde derivates in plasma. Therefore, LA showed antioxidant properties that may have beneficial effects under diabetes. Such results may represent LA as one of the plant components in the development of new drugs that will correct metabolic and functional disorders in leukocytes under diabetes.

Effect of Non-Surgical Periodontal Treatment on Oxidative Stress Markers in Leukocytes and Their Interaction with the Endothelium in Obese Subjects with Periodontitis: A Pilot Study.

Aim: The primary objective of this pilot study was to evaluate the effect of non-surgical periodontal treatment. The secondary aim was to evaluate the effect of dietary therapy on both parameters of oxidative stress in leukocytes and leukocyte-endothelial cell interactions in an obese population. Methods: This was a pilot study with a before-and-after design. Forty-nine obese subjects with periodontitis were randomized by means of the minimization method and assigned to one of two groups, one of which underwent dietary therapy while the other did not. All the subjects underwent non-surgical periodontal treatment. We determined periodontal, inflammatory and oxidative stress parameters—total reactive oxygen species (ROS), superoxide production, intracellular Ca2+, mitochondrial membrane potential and superoxide dismutase (SOD) activity. We also evaluated interactions between leukocytes and endothelium cells—velocity, rolling flux and adhesion—at baseline and 12 weeks after intervention. Results: Periodontal treatment improved the periodontal health of all the patients, with a reduction in serum retinol-binding protein 4 (RBP4), total superoxide production and cytosolic Ca2+ in leukocytes. In the patients undergoing dietary therapy, there were less leukocyte adhesion to the endothelium, an effect that was accompanied by a decrease in TNFα, P-selectin and total ROS and an increase in SOD activity. Conclusions: Whereas non-surgical periodontal treatment induces an improvement in leukocyte homeostasis, dietary therapy as an adjuvant reduces systemic inflammation and increases antioxidant status which, in turn, modulates leukocyte-endothelium dynamics.

Changes in redox and endoplasmic reticulum homeostasis are related to congenital generalized lipodystrophy type 2

CGL type 2 is a rare autosomal recessive syndrome characterized by an almost complete lack of body fat. CGL is caused by loss-of-function mutations in both alleles of the BSCL2 gene that codifies to seipin. Subjects often show hyperglycemia, decreased HDL-c, and hypoadiponectinemia. These laboratory findings are important triggers for changes in redox and ER homeostasis. Therefore, our aim was to investigate whether these intracellular mechanisms are associated with this syndrome. We collected blood from people from Northeastern Brazil with 0, 1, and 2 mutant alleles for the rs786205071 in the BSCL2 gene. Through the qPCR technique, we evaluated the expression of genes responsible for triggering the antioxidant response, DNA repair, and ER stress in leukocytes. Colorimetric tests were applied to quantify lipid peroxidation and to evaluate the redox status of glutathione, as well as to access the panorama of energy metabolism. Long extension PCR was performed to observe leukocyte mitochondrial DNA lesions, and the immunoblot technique to investigate plasma adiponectin concentrations. Subjects with the rs786205071 in both BSCL2 alleles showed increased transcription of NFE2L2, APEX1, and OGG1 in leukocytes, as well as high concentrations of malondialdehyde and the GSSG:GSH ratio in plasma. We also observed increase of mitochondrial DNA lesions and XBP1 splicing, as well as a decrease in adiponectin and HDL-c. Our data suggest the presence of lipid lesions due to changes in redox homeostasis in that group, associated with increased levels of mitochondrial DNA damage and transcriptional activation of genes involved with antioxidant response and DNA repair.

Attenuated flow-induced dilatation of middle cerebral arteries is related to increased vascular oxidative stress in rats on a short-term high salt diet.

Recent studies have shown that high salt (HS) intake leads to endothelial dysfunction and impaired vascular reactivity in different vascular beds in both animal and human models, due to increased oxidative stress. The objective of this study was to assess vascular response to flow-induced dilatation (FID) and to elucidate the role of vascular oxidative stress/antioxidative capacity in middle cerebral arteries (MCAs) of HS-fed rats in vitro. The novelty of this study is in demonstrating impaired flow-induced dilatation of MCAs and down-regulation of vascular antioxidant genes with HS intake, leading to increased levels of oxidative stress in blood vessels and peripheral lymph organs, which together contribute to impaired FID. In addition, results show increased oxidative stress in leukocytes of peripheral lymph organs, suggesting the occurrence of inflammatory processes due to HS intake. Recirculation of leukocytes might additionally increase vascular oxidative stress in vivo. The aim of this study was to determine flow-induced dilatation (FID) and the role of oxidative stress/antioxidative capacity in isolated, pressurized middle cerebral arteries (MCAs) of high salt (HS)-fed rats. Healthy male Sprague-Dawley rats (11weeks old) were fed low salt (0.4% NaCl; LS group) or high salt (4% NaCl; HS group) diets for 1week. Reactivity of MCAs in response to stepwise increases in pressure gradient (Δ10-Δ100mmHg) was determined in the absence or presence of the superoxide dismutase (SOD) mimetic TEMPOL and/or the nitric oxide synthases (NOS) inhibitor N(ω) -nitro-l-arginine methyl ester (l-NAME). mRNA levels of antioxidative enzymes, NAPDH-oxidase components, inducible (iNOS) and endothelial nitric oxide synthases (eNOS) were determined by quantitative real-time PCR. Blood pressure (BP), antioxidant enzymes activity, oxidative stress in peripheral leukocytes, lipid peroxidation products and the antioxidant capacity of plasma were measured for both groups. FID was reduced in the HS group compared to the LS group. The presence of TEMPOL restored dilatation in the HS group, with no effect in the LS group. Expression of glutathione peroxidase 4 (GPx4) and iNOS in the HS group was significantly decreased; oxidative stress was significantly higher in the HS group compared to the LS group. HS intake significantly induced basal reactive oxygen species production in the leukocytes of mesenteric lymph nodes and splenocytes, and intracellular production after stimulation in peripheral lymph nodes. Antioxidant enzyme activity and BP were not affected by HS diet. Low GPx4 expression, increased superoxide production in leukocytes, and decreased iNOS expression are likely to underlie increased oxidative stress and reduced nitric oxide bioavailability, leading to impairment of FID in the HS group without changes in BP values.

Effects of Sublethal Doses of Lampcon and Saturn Pesticides on Some Clinicopathological and Immunological Parameters in Rats

This study aimed to evaluate the clinicopathological and immunological profiles of two pesticides widely used in Egypt, Lampcon and Saturn in rats. Thirty male Sprague Dawley rats divided into three groups each of 10 were used in this study. The first group received no treatment and served as control, the second group received Lampcon at a dose of 11.2 mg/kg body weight, and the third group received Saturn at a dose of 282 mg/kg body weight. The treatments were administrated daily via food for 60 successive days. Blood samples were collected for evaluating the selected hematological, biochemical and immunological parameters. The results of hematological variables implicated no significant effect on red blood cell parameters, but there was a significant leukopenia, lymphopenia and neutrophilia in both treated groups. The blood biochemical parameters showed a significant increase in serum levels of urea and enzymatic activities of ALT, AST and ALP. The findings of immunological studies clarified that Lampcon and Saturn induced oxidative stress in leukocytes and had immunosuppressive effects. The main histopathological alterations induced by Lampcon and Saturn included lymphoid depletion in lymphoid organs. Conclusion: The results from the study have shown that exposure of rats to Lampcon and Saturn pesticides pose many hematological, biochemical, and histopathological alterations in addition to immunotoxic effects. It is thus advisable that Lampcon and Saturn should be used with caution and at the recommended levels indicated by the producing company to ensure minimal exposure to these pesticides during domestic, veterinary, agricultural or industrial use.

Pro-inflammatory cytokines and leukocyte oxidative burst in chronic kidney disease: culprits or innocent bystanders?

Pro-inflammatory cytokines are elevated in chronic kidney disease (CKD), a condition characterized by microinflammation with oxidative stress as key feature. However, their role in the inflammatory response at uraemic concentrations has not yet been defined. In this study, the contribution of cytokines on induction of leukocyte oxidative stress was investigated. Whole blood from healthy donors was incubated with 20-1400 pg/mL TNFα, 5-102.8 pg/mL IL-6, 20-400 pg/mL IL-1β and 75-1200 pg/mL IL-18 separately or in combination. Oxidative burst was measured, at baseline and after stimulation with fMLP (Phagoburst™). The effect of the TNFα blocker, adalimumab (Ada), was evaluated on TNFα-induced ROS production. Finally, the association between TNFα and the composite end point all-cause mortality or first cardiovascular event was analysed in a CKD population stage 4-5 (n = 121). While interleukin (IL)-6, IL-1β and IL-18 alone induced no ROS activation of normal leukocytes, irrespective of concentrations, TNFα induced ROS activation at baseline (P < 0.01) and after fMLP stimulation (P < 0.05), but only at uraemic concentrations in the high range (400 and 1400 pg/mL). A similar pattern was observed with all cytokines in combination, but already at intermediate uraemic concentrations (all P < 0.05, except for monocytes after fMLP stimulation: n.s.), suggesting synergism between cytokines. ROS production induced by TNFα (400 pg/mL) and the cytokine combination was blocked with Ada. Uraemia-related oxidative stress in leukocytes of haemodialysis patients was however not blocked by Ada. In patients, TNFα was not associated to adverse events (HR: 1.52, 95% CI 0.81-2.85, P = 0.13). Among several pro-inflammatory cytokines, TNFα alone was pro-oxidative but only at high-range uraemic concentrations. Adding a TNFα blocker, Ada, blocked this ROS production, but not the oxidative stress in blood samples from haemodialysis patients, suggesting that other uraemic toxins than TNFα are more crucial in this process. However, the lack of association between TNFα and mortality suggests that the role of TNFα-linked oxidative stress is limited.

Altered Mitochondrial Function and Oxidative Stress in Leukocytes of Anorexia Nervosa Patients

ContextAnorexia nervosa is a common illness among adolescents and is characterised by oxidative stress.ObjectiveThe effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated.Design and settingA multi-centre, cross-sectional case-control study was performed.PatientsOur study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women.Main outcome measuresAnthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells.ResultsMitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (P<0.05), mitochondrial membrane potential (P<0.01) and GSH levels (P<0.05), and an increase in ROS production (P<0.05) with respect to control subjects. Furthermore, a reduction of mitochondrial mass was detected in leukocytes of the anorexic patients (P<0.05), while the activity of mitochondrial complex I (P<0.001), but not that of complex III, was found to be inhibited in the same population.ConclusionsOxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

Is glycemic control modulating endoplasmic reticulum stress in leukocytes of type 2 diabetic patients?

Oxidative and endoplasmic reticulum (ER) stress is related to type 2 diabetes (T2D), but the influence of glycemic control on these parameters and its relationship with leukocyte-endothelial interactions is not known. In our study population consisting of 164 diabetic patients, (102 with glycated hemoglobin [HbA1c] <7% and 62 with HbA1c >7%) and 84 nondiabetic controls, we have verified a common anthropometric and metabolic pattern of T2D with dyslipidemia. Inflammatory parameters (high-sensitive C-reactive protein [hs-CRP] and tumor necrosis factor alpha [TNFα]) and E-selectin levels were enhanced in the HbA1c >7% group with regard to controls. O2 consumption and mitochondrial membrane potential were lower in diabetic patients than in controls. Reactive oxygen species (ROS) production was enhanced in diabetic patients than in controls and positively correlated with HbA1c levels. GRP78 levels were higher in both diabetic groups. However, HbA1c <7% patients displayed higher levels of spliced X-box binding protein 1 (sXBP1), whereas HbA1c >7% patients exhibited preferentially enhanced levels of CHOP (CCAAT/enhancer binding protein [C/EBP] homologous protein) and activating transcription factor 6 (ATF6). Reduced leukocyte rolling velocity and increased rolling flux and adhesion were observed in diabetic patients. Our findings lead to the hypothesis of an association between poor glycemic control in T2D and increased leukocyte ROS production and chronic ER stress that could finally promote leukocyte-endothelial interactions, which, in turn, poses a risk of vascular complications for these patients.

Mitochondrial Impairment and Oxidative Stress in Leukocytes after Testosterone Administration to Female-To-Male Transsexuals

Testosterone undecanoate (T) treatment is common in female-to-male transsexuals (FtMs) but can induce impairment of mitochondrial function and oxidative stress. The effect of T treatment on the mitochondrial function and redox state of leukocytes of FtMs subjects was evaluated. This was an observational study conducted in a university hospital. Fifty-seven FtMs were treated with T (1,000 mg) for 12 weeks, after which anthropometric and metabolic parameters and mitochondrial function were evaluated. Anthropometric and metabolic parameters were evaluated. Mitochondrial function was studied by assessing mitochondrial oxygen (O2) consumption, membrane potential, reactive oxygen species (ROS) production, glutathione levels (GSH), and the reduced glutathione/oxidized glutathione (GSH)/(GSSG) ratio in polymorphonuclear cells. T treatment led to mitochondrial impairment in FtMs as a result of a decrease in mitochondria O2 consumption, the membrane potential, GSH levels, and the (GSH)/(GSSG) ratio and an increase in ROS production. Mitochondrial O2 consumption and membrane potential negatively correlated with T levels, which was further confirmed that the T treatment had induced mitochondrial dysfunction. T also produced a significant increase in total testosterone, free androgenic index, and atherogenic index of plasma, and a decrease in sex hormone-binding globulin and high-density lipoprotein cholesterol. Treatment of FtMs with T can induce impairment of mitochondrial function and a state of oxidative stress. This effect should be taken into account in order to modulate possible comorbidities in these patients.

Uremia-Related Oxidative Stress in Leukocytes Is Not Triggered by β2-Microglobulin

Chronic kidney disease (CKD) is characterized by low-grade inflammation and increased risk for cardiovascular disease. The interest in β2-microglobulin (B2M) as a marker for cardiovascular outcome with and without CKD has grown. Clinical studies suggested that B2M could be involved in the pathogenesis of vascular disease, for which chronic leukocyte activation is a pathogenic factor. We investigated whether B2M is proinflammatory by inducing oxidative burst in leukocytes. Oxidative burst was measured at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP), Escherichia coli, or phorbol-12-myristate-acetate (PMA) in the whole blood of healthy volunteers in the absence (saline) and presence of human B2M (hB2M; 10 and 50mg/L) versus uremic whole blood. Because of suspicion of contamination, hB2M was dialyzed for purification and purified B2M (dB2M) and dialysates were tested in the burst test. As a comparator, reactive oxygen species (ROS) in response to lipopolysaccharide (LPS) was measured. Unpurified hB2M strongly enhanced ROS in monocytes and granulocytes after E. coli and PMA and moderately after fMLP stimulation compared with control (P < .01) and uremia (P < .01) whereas at baseline hB2M only induced ROS in granulocytes (P < .05). After purification, dB2M no longer increased burst activity, suggesting that contamination was responsible for the initial effect. An endotoxin concentration of less than 1.5 EU/mL, as observed in hB2M, could not induce oxidative stress. This study suggests that B2M, a traditional marker for middle molecule retention and a novel marker for cardiovascular outcome, may not by itself cause vascular damage by influencing inflammatory response due to induction of leukocyte free radical production. However, an effect on other cell types involved cannot be excluded. Our data further reveal that this type of research might be skewed by non-LPS contaminants, and that care should be taken to exclude this bias.

Assessment of oxidative stress in leukocytes and granulocyte function following oral administration of a silibinin-phosphatidylcholine complex in cats

To determine the effect of oral administration of a silibinin-phosphatidylcholine complex (SPC) on oxidative stress in leukocytes and granulocyte function in healthy cats. 10 purpose-bred adult cats. Cats were administered SPC (10 mg/kg/d) orally for 5 days; blood samples were collected prior to and immediately after the 5-day treatment period. Leukocytes were incubated with monochlorobimane for detection of reduced glutathione (GSH) via flow cytometry. Leukocytes were also incubated with dihydrorhodamine 123 and mixed with Escherichia coli conjugated to a fluorescent marker to measure E coli phagocytosis and the subsequent oxidative burst via flow cytometry. Activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, along with the reduced glutathione-to-oxidized glutathione (GSH:GSSG) ratio and a measure of lipid peroxidation (malondialdehyde concentration [micromol/L of blood]), were measured spectrophotometrically. The mean fluorescence intensity (MFI), representing GSH content, increased significantly in feline lymphocytes and granulocytes following 5 days of oral administration of SPC. Mean +/- SD lymphocyte MFI significantly increased from 27.8 +/- 9.0 to 39.6 +/- 6.7, and the granulocyte MFI increased from 508.6 +/- 135.6 to 612.1 +/- 122.9. Following 5 days of SPC administration, the percentage of phagocytic cells that were responding optimally significantly increased (from 37 +/- 11.8% to 45 +/- 17.5%). Other measures of oxidative stress did not change significantly. In cats, oral administration of supplemental SPC appears to increase granulocyte GSH content and phagocytic function, both of which would be potentially beneficial in cats with diseases associated with oxidative stress.

Deficient heat shock protein 70 response to stress in leukocytes at onset of type 1 diabetes

Type 1 diabetes is caused by the immune-mediated destruction of pancreatic beta cells. Animal models of the disease demonstrate an increased susceptibility of beta cells to immunological attacks due to their defective stress-responsiveness. To investigate the stress-responsiveness in human type 1 diabetes we analyzed the heat-inducibility of the dominant stress protein heat shock protein (Hsp)70 in diabetic patients at different disease stages. At diabetes-manifestation heat-induced Hsp70 levels in peripheral blood mononuclear cells (PBMC) reached only about 25% of the levels expressed by heat-treated PBMC from non-diabetic subjects ( p < 0.05). Heat-responsiveness improved with disease duration and was re-established at more than eight months after disease-manifestation. Hyperthermia-induced Hsp70 expression was decreased by the T-helper 1-associated cytokine interferon-γ and increased by the T-helper 2-associated transforming growth factor-β. We conclude that impaired cellular stress-responsiveness, aggravated by the inflammatory milieu at the onset of type 1 diabetes, contributes to disease manifestation.

Oxidative Stress in Mononuclear Cells Plays a Dominant Role in Their Adhesion to Mouse Femoral Artery After Injury

Leukocyte recruitment plays a pivotal role during inflammation after vascular injury. The importance of oxidative stress in vascular injury and its modulation by angiotensin II receptor blockers (olmesartan) have been demonstrated. We examined the contribution of leukocyte-associated oxidative stress in acute-phase leukocyte recruitment and its modulation by olmesartan. Male mice were treated with olmesartan (5 mg/kg per day) or vehicle for 7 days before the transluminal wire injury of the femoral artery. Intravital microscopy of the artery revealed that the mechanical injury increased adherent leukocytes at both 24 hours and 7 days after the injury, which was significantly reduced by olmesartan treatment. Dihydroethidium-associated fluorescence intensity observed in vehicle-treated mice was significantly diminished under olmesartan treatment. Apocynin, a nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, showed a similar inhibitory effect on the leukocyte adhesion. Adoptive transfer of mononuclear cells, harvested from mice after wire injury, but not from those without wire injury, exhibited adhesion to the recipient injured artery. Furthermore, olmesartan treatment of mononuclear cells, but not of injured vasculature, reduced their recruitment to the injured artery. These data indicate that leukocyte recruitment to the mechanically injured artery is mediated by oxidative stress in leukocytes but not in vasculatures. Treatment with olmesartan blocked leukocyte recruitment by antagonizing mononuclear cells-associated oxidative stress.

Conservation of IL‐6 trans‐signaling mechanisms controlling L‐selectin adhesion by fever‐range thermal stress

Fever is associated with improved survival during infection in endothermic and ectothermic species although the protective mechanisms are largely undefined. Previous studies indicate that fever-range thermal stress increases the binding activity of the L-selectin homing receptor in human or mouse leukocytes, thereby promoting trafficking to lymphoid tissues across high endothelial venules (HEV). Here, we examined the evolutionary conservation of thermal regulation of L-selectin-like adhesion. Leukocytes from animals representing four taxa of vertebrates (mammals, avians, amphibians, teleosts) were shown to mediate L-selectin-like adhesion under shear to MECA-79-reactive ligands on mouse HEV in cross-species in vitro adherence assays. L-selectin-like binding activity was markedly increased by fever-range thermal stress in leukocytes of all species examined. Comparable increases in L-selectin-like adhesion were induced by thermal stress, IL-6, or the IL-6/soluble IL-6 receptor fusion protein, hyper-IL-6. Analysis of the molecular basis of thermal regulation of L-selectin-like adhesion identified a common IL-6 trans-signaling mechanism in endotherms and ectotherms that resulted in activation of JAK/STAT signaling and was inhibited by IL-6 neutralizing antibodies or recombinant soluble gp130. Conservation of IL-6-dependent mechanisms controlling L-selectin adhesion over hundreds of millions of years of vertebrate evolution strongly suggests that this is a beneficial focal point regulating immune surveillance during febrile inflammatory responses.

Oxidative stress in leukocytes from young prematurely aging mice is reversed by supplementation with biscuits rich in antioxidants

Aging is associated with a progressive dysregulation of immune responses as a result of increased oxidative stress. Therefore, we have assessed the oxidative stress status of peritoneal leukocytes from young prematurely aging mice (PAM) as compared with non-prematurely aging mice (NPAM), as well as the effects on this oxidative stress of a dietary supplementation with biscuits rich in antioxidants (vitamin C, vitamin E, β-carotenes, zinc and selenium). We found that, in the peritoneal leukocytes, the levels of several parameters of oxidation such as extracellular superoxide anion ( O 2 - ) , Prostaglandin E 2 (PGE 2), nitric oxide, oxidized glutathione (GSSG) and lipid peroxidation (malondialdehyde, MDA) were higher in PAM as compared with NPAM, whereas the antioxidant defences such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities, as well as reduced glutathione (GSH) levels, were decreased. Consequently, young PAM showed an oxidative stress in their leukocytes, which is characteristic of mice of an older chronological age. Antioxidant diet supplementation was able to restore redox homeostasis, increasing the antioxidant and decreasing the oxidant levels. Accordingly, supplementation with adequate levels of antioxidants, from an early age, could be useful to preserve health, especially in prematurely aging populations.