Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells.

Myeloperoxidase (MPO) has been reported in prostate tissue, and considering its pro-oxidant properties, this location might be linked to prostate pathology. The possibility that the glandular prostatic tissue might be the source of MPO and its potential inflammatory effects must be tested. Human prostate material was obtained from prostate biopsies and radical prostatectomies. Immunohistochemistry was performed using MPO-specific human antibody. In situ hybridization using MPO-specific probes and laser-assisted microdissection for quantitative real-time RT-PCR were performed to observe whether MPO is being produced in prostate tissue. Mass spectrometry on prostate biopsies was used to detect products of MPO activity in nucleic acids (DNA/RNA). MPO contribution to intracellular accumulation of ROS and interleukin-8 in prostatic epithelial cells was monitored in vitro. Immunohistochemistry confirmed cellular localization of MPO in epithelial cells of the prostate. The staining varied from light to high intensity. In situ hybridization did not address the presence of mRNA coding for MPO. No MPO-specific modifications on nucleic acids were detected. Mox-LDL was a major factor inducing ROS and cytokines production in prostatic epithelial cells. We did not demonstrate that MPO was synthetized by prostatic epithelial cells. However, in vitro experiments showed the ability of MPO to potentiate the ROS production and inflammation on prostate epithelial cells. Results do not allow us to demonstrate a role of MPO in prostate to date but further studies are mandatory to focus on the potential impact of MPO in the development of prostatic diseases.

Quantification of stromal reaction in breast carcinoma and its correlation with tumor grade and free progression survival.

Cancer progression results from a complex interplay between tumor cells and the extracellular milieu. In breast carcinoma, the stromal microenvironment has been suggested to play a major role in promoting tumor growth, progression, and invasion. The stroma of 154 resected specimens of invasive breast carcinoma of no special type was quantified using a digital image analyzer. Statistical analyses were performed between the quantity of stroma and survival, as well as between progression-free survival and clinicopathological data. Levels of myofibroblastic stroma varied from 0-46%, with a median of 15.1% and a standard deviation of 7.5. The myofibroblastic stromal reaction was statistically greater in grade 2 and 3 tumors (p = 0.029). Furthermore, there was a trend for worse progression-free survival in the group of node-negative tumors with strong smooth-muscle actin stromal expression (Log rank = 0.075). The present study demonstrates that the myofibroblastic reaction of breast invasive carcinoma of no special type is not merely a passive reaction, but seems to be an integral part of the neoplastic process by facilitating tumor progression and invasion. Additional, larger studies on mechanisms of stromal change are needed and may potentially lead to novel treatments.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

A prospective clinical study of the implications of IL-8 in the diagnosis, aggressiveness and prognosis of prostate cancer.

AIM: We evaluated the relationship between IL-8 and prostate cancer (PCa) with emphasis on diagnosis, aggressiveness and prognosis. MATERIALS & METHODS: Prostate-specific antigen (PSA) and serum IL-8 were collected from patients undergoing prostate biopsy. IL-8 expression was evaluated on immunohistochemistry with IL-8 labeling index. Complete follow-up of this cohort was achieved over a period of up to 6 years with continuous follow-up of PSA levels. RESULTS: Among 135 patients, serum IL-8 level did not correlate to the diagnosis or aggressiveness of PCa. In 52 radical prostatectomy specimens, a higher IL-8 labeling index was detected in the tumor areas (0.4 ± 0.2 vs 0.33 ± 0.2; p = 0,007) but did not correlate to any of the prognostic markers: D'Amico classification (p = 0.52), Gleason score (p = 0.45), perineural (p = 0.83) and capsular invasion (p = 0.75). No correlation was found to PSA biochemical-free failure. CONCLUSION: IL-8 serum level was not a significant predictor of diagnosis, aggressiveness or prognosis of PCa.

Relationship between oxidative stress and erectile function.

The aim of this study was to investigate markers of inflammation and oxidative stress in the corpus cavernosum (CC) and to compare levels of inflammatory markers recorded in CC to venous blood from the arm to examine the potential impact of inflammatory parameters on erectile function and endothelial dysfunction in vitro. Ninety-seven patients with no complaint of erectile dysfunction (ED) at inclusion were prospectively included and completed the Erectile Function domain of the IIEF questionnaire. Several parameters, including lipids, MPO-dependent oxidised LDL (Mox-LDL), IL-8, IL-18, were measured. After RNA extraction, the expression of eNOS was analysed. A paired t-test was used for comparisons between arm and CC blood results. A two-way ANOVA was used to estimate the effects of IL-18 and IL-8 on the IIEF score. Mean patient age was 59 ± 14.5 years. IL-18, Mox-LDL, and Mox-LDL/ApoB levels were significantly increased in CC compared to arm blood. The IIEF score was correlated with IL-18 levels in the venous blood (R = -0.31, p = .003) and in the CC (R = -0.37, p = .004) and with IL-8 (R = -0.31, p = .009 and R = -0.28, respectively, p = .02). There was a significant effect with the IL-18 on IIEF potentiated by high serum IL-8 concentrations. IL-18 and Mox-LDL significantly decreased eNOS mRNA expression in human aortic endothelial cell line (HAEC). These preliminary results address the importance of inflammation in the CC and highlight a difference in marker concentrations between venous and CC blood. However, they do not show any difference in terms of clinical erectile score predictivity. Involvement of inflammatory cytokines isolated in CC in the genesis of ED requires further studies.

Leukocyte phosphodiesterase expression after lipopolysaccharide and during sepsis and its relationship with HLA-DR expression.

Phosphodiesterases (PDEs) may modulate inflammatory pathways, but PDE expression is poorly documented in humans with sepsis. Using quantitative PCR on whole blood leukocytes, we characterized PDE mRNA expression in healthy volunteers (n = 20), healthy volunteers given lipopolysaccharide (LPS; n = 18), and critically ill patients with (n = 20) and without (n = 20) sepsis. PDE4B protein expression was also studied in magnetic-activated cell sorting (MACS)-isolated CD15+ neutrophils (from 7 healthy volunteers, 5 patients without and 5 with sepsis). We studied relationships between PDE expression, HLA-DR (mRNA and expression on CD14+ monocytes), tumor necrosis factor (TNF)-α, and interleukin (IL)-10 levels. LPS administration in volunteers was associated with increases in PDE4B and PDE4D and decreases in PDE4A and PDE7A mRNAs. The observed global down-regulation of the HLA-DR complex was correlated with PDE7A. Critically ill patients had lower TNF-α/IL-10 mRNA ratios than the volunteers had and global down-regulation of the HLA-DR complex. Septic patients had persistently lower mRNA levels of PDE7A, PDE4A, and 4B (also at a protein level) and decreasing levels of PDE4D over time. Low PDE4D mRNA levels correlated negatively with HLA-DMA and HLA-DMB. LPS administration and sepsis are, therefore, associated with different PDE mRNA expression patterns. The effect of PDE changes on immune dysfunction and HLA-DR expression requires further investigation.

Nuclear localization of glutamate-cysteine ligase is associated with proliferation in head and neck squamous cell carcinoma.

Glutathione (GSH) is the keystone of the cellular response toward oxidative stress. Elevated GSH content correlates with increased resistance to chemotherapy and radiotherapy of head and neck (HN) tumors. The purpose of the present cross-sectional study was to evaluate whether the expression of glutamate-cysteine ligase (GCL) accounts for the increased GSH availability observed in HN squamous cell carcinoma (SCC). For that purpose, the messenger (m)RNA levels of the modifier (M) and catalytic (C) subunits of GCL and its putative regulators (namely, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) were monitored in 35 surgical resections of untreated HNSCC. The localization of GCLM was evaluated using in situ hybridization and immunohistochemistry. GCLM expression was significantly increased in tumor samples, compared with normal mucosa, both at the mRNA and protein level (P=0.029), but the pathway of GCLM activation remains to be elucidated. Protein expression of GCLM was detected in the cytoplasm and nucleus. GCLM and the proliferation marker Ki-67 displayed a similar distribution, being both mainly expressed at the periphery of tumor lobules. The present study reported increased expression of GCL and the rate-limiting enzyme of GSH synthesis, within HNSCC. The nuclear localization of GCLM and the concomitant expression of Ki-67 suggested that the localization of GSH synthesis contributes to the protection against oxidative stress within hotspots of cell proliferation.

Tumour-associated osteomalacia and hypoglycaemia in a patient with prostate cancer: is Klotho involved?

Tumour-associated osteomalacia is a paraneoplastic syndrome caused by renal phosphate wasting, leading to severe hypophosphataemia. Excess of circulating fibroblast growth factor 23 (FGF23) is the likely cause, acting via the FGF23/α-Klotho coreceptor, a critical regulator of phosphate metabolism. The other possible effects of that complex in humans are still under investigation. We present a case of an 84-year-old Belgian man, presenting prostate cancer with bone metastases. From June 2010 to March 2013, he presented three episodes of disease progression. From January 2012, the patient developed a progressively marked dorsal kyphosis with significant hypophosphataemia. The calculated TRP (tubular reabsorption of phosphate) was decreased and the FGF23 increased. Mid-March 2013, the patient died after a profound unconsciousness due to hypoglycaemia with hypothermia. We hypothesised that the two paraneoplastic manifestations of this patient (tumour-associated osteomalacia and refractory hypoglycaemia) were due to one cause chain with two main nodes-FGF23 and its coreceptor Klotho..

Inhibition of myeloperoxidase activity by the alkaloids of Peganum harmala L. (Zygophyllaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds and aerial parts of Peganum harmala L. are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure ß-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant. MATERIALS AND METHODS: MPO inhibition was tested using taurine chloramine test. The inhibition of LDL oxidation induced by MPO was carried out. The molecular docking analysis of Peganum harmala alkaloids on MPO was performed using the Glide XP docking protocol and scoring function and the redox potential of alkaloids was determined using an Epsilon potentiostat. The concentration of harmala alkaloids was determined using HPLC analysis. RESULTS: The HPLC profiling of the active total alkaloids indicates that ß-carboline e.g. harmine, harmaline, harmane, harmol and harmalol are major components. As ß-carbolines resemble tryptamine, of which derivatives are efficient inhibitors of MPO, the harmala alkaloids were tested for their activity on this enzyme. Total alkaloids of the seeds and of the aerial parts strongly inhibited MPO at 20µg/mL (97±5% and 43±4%, respectively) whereas, at the same concentration, those of the roots showed very low inhibition (15±6%). Harmine, harmaline and harmane demonstrated a significant inhibition of MPO at IC50 of 0.26, 0.08 and 0.72µM respectively. These alkaloids exerted a similar inhibition effects on MPO-induced LDL oxidation. Molecular docking analysis of Peganum harmala alkaloids on MPO showed that all active Peganum harmala alkaloids have a high affinity on the active site of MPO (predicted free energies of binding up to -3.1kcal/mol). Measurement of redox potentials versus the normal hydrogen electrode clearly differentiated (i) the high MPO inhibitory activity of harmine, harmaline and harmane (+1014, 1014 and 1003mV, respectively); and (ii) the low activity of harmalol and harmol (+629/778 and 532/644mV, respectively). A reverse phase HPLC method has been developed to determine simultaneously five alkaloids of Peganum harmala. Seeds contained all five ß-carboline derivatives with the main active alkaloids, harmaline and harmine, being up to 3.8% and 2.9%, respectively. Up to 3.2% of harmine was determined in the roots. The four ß-carboline derivatives, harmine, harmaline, harmane and harmalol were identified in the aerial parts. The highest inhibitory effect observed in seeds and the moderate effect of aerial parts could be explained by their harmine and harmaline content. In contrast, the very weak inhibition of the root extract, despite the presence of harmine, may tentatively be explained by the high concentration of harmol which can reduce Compound II of MPO to the native form. CONCLUSION: The inhibition of MPO by Peganum harmala ß-carboline alkaloids, herein reported for the first time, may explain the anti-inflammatory effect traditionally attributed to its herbal medicine.

Hybrid molecules inhibiting myeloperoxidase activity and serotonin reuptake: a possible new approach of major depressive disorders with inflammatory syndrome.

OBJECTIVES: Major depressive disorder (MDD) is accompanied with an imbalance in the immune system and cardiovascular impairments, such as atherosclerosis. Several mechanisms have been pointed out to underlie this rather unexpected association, and among them the activity of myeloperoxidase (MPO). The aim of our study was to find compounds that inhibit both MPO and serotonin transporter (SERT) for treating MDD associated with cardiovascular diseases. METHODS: SERT inhibition was assessed with measuring of [(3) H]-serotonin uptake using HEK-293 MSR cells. MPO inhibition was determined by taurine chloramine test on 3-(aminoalkyl)-5-fluoroindole derivatives and on clinically relevant antidepressants. All kinetic measurements were performed using a temperature-controlled stopped-flow apparatus (model SX-18 MV). Promising lead compounds were docked onto SERT 3D structure modelled using the LeuT structure complexed to tryptophan (PDB code 3F3A). Their toxicological profile was also assessed. KEY FINDINGS: 3-(aminoalkyl)-5-fluoroindole derivative with 5 carbons on the side chain and paroxetine showed the best activity on both MPO and SERT at the nanomolar range. Paroxetine was found to be the first irreversible MPO inhibitor at nanomolar concentrations. CONCLUSIONS: Our results put forward the first hybrid molecule (compound 25) and drug (paroxetine) that can be especially used in MDD associated with inflammatory syndrome.

Characterization and antioxidant properties of six Algerian propolis extracts: ethyl acetate extracts inhibit myeloperoxidase activity.

Because propolis contains many types of antioxidant compounds such as polyphenols and flavonoids, it can be useful in preventing oxidative damages. Ethyl acetate extracts of propolis from several Algerian regions show high activity by scavenging free radicals, preventing lipid peroxidation and inhibiting myeloperoxidase (MPO). By fractioning and assaying ethyl acetate extracts, it was observed that both polyphenols and flavonoids contribute to these activities. A correlation was observed between the polyphenol content and the MPO inhibition. However, it seems that kaempferol, a flavonoid, contributes mainly to the MPO inhibition. This molecule is in a high amount in the ethyl acetate extract and demonstrates the best efficiency towards the enzyme with an inhibiting concentration at 50% of 4 ± 2 µM.

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100.

Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.

Myeloperoxidase-dependent LDL modifications in bloodstream are mainly predicted by angiotensin II, adiponectin, and myeloperoxidase activity: a cross-sectional study in men.

The present paradigm of atherogenesis proposes that low density lipoproteins (LDLs) are trapped in subendothelial space of the vascular wall where they are oxidized. Previously, we showed that oxidation is not restricted to the subendothelial location. Myeloperoxidase (MPO), an enzyme secreted by neutrophils and macrophages, can modify LDL (Mox-LDL) at the surface of endothelial cells. In addition we observed that the activation of the endothelial cells by angiotensin II amplifies this process. We suggested that induction of the NADPH oxidase complex was a major step in the oxidative process. Based on these data, we asked whether there was an independent association, in 121 patients, between NADPH oxidase modulators, such as angiotensin II, adiponectin, and levels of circulating Mox-LDL. Our observations suggest that the combination of blood angiotensin II, MPO activity, and adiponectin explains, at least partially, serum Mox-LDL levels.

Increased basal and alum-induced interleukin-6 levels in geriatric patients are associated with cardiovascular morbidity.

BACKGROUND/AIM OF THE STUDY: Low-grade systemic inflammation was suggested to participate to the decline of physiological functions and increased vulnerability encountered in older patients. Geriatric syndromes encompass various features such as functional dependence, polymorbidity, depression and malnutrition. There is a strong prevalence of cardiovascular diseases and related risk factors and chronic cytomegalovirus infections in the geriatric population. As these underlying conditions were proposed to influence the inflammatory state, the aim of this study was to assess their potential contribution to the association of geriatric syndromes with inflammatory parameters. METHODOLOGY: We recruited 100 subjects in the general population or hospitalized for chronic medical conditions (age, 23-96 years). We collected information on clinical status (medical history, ongoing comorbidities, treatments and geriatric scales), biological parameters (hematological tests, cytomegalovirus serology) and cytokines production (basal and alum-induced interleukin (IL)-1ß and IL-6 levels). Using stepwise backward multivariate analyses, we defined which set of clinical and biological variables could be predictive for increased inflammatory markers. PRINCIPAL FINDINGS: We confirmed the age-associated increase of circulating IL-6 levels. In contrast to geriatric scales, we found history of cardiovascular diseases to be strongly associated for this parameter as for high IL-6 production upon ex vivo stimulation with alum. CONCLUSIONS: Association between low-grade inflammation and geriatric conditions could be linked to underlying cardiovascular diseases.

Frailty in old age is associated with decreased interleukin-12/23 production in response to toll-like receptor ligation.

Aging is associated with progressive alterations of immune functions, leading to higher susceptibility to bacterial and viral infections and reduced vaccine responses. Data concerning cytokine production in response to Toll-like receptor (TLR) ligands are highly variable in old people, reflecting the heterogeneity of the geriatric population. The aim of our study was to define the relative contribution of age and clinical status on TLR-induced interleukin (IL)-12p70 and IL-23 production as these cytokines play an important role in the protection against intracellular and extracellular pathogens, respectively. For this purpose, we recruited 100 subjects (aged 23-96 years) in the general population or hospitalized for chronic diseases. We collected information on clinical status (medical history, ongoing comorbidities, treatments and geriatric scales), biological parameters (biochemical and hematological tests, telomere length determination, cytomegalovirus serology). Whole blood samples were stimulated with a combination of TLR4 and TLR7/8 ligands. We performed univariate and stepwise backward multivariate analyses regression to define which set of clinical variables could be predictive for IL-12p70 and IL-23 production in these conditions. Our results indicated that age was not correlated with TLR-mediated IL-12p70 and IL-23 production. In contrast, poor nutritional status and frailty in subjects >75 years were associated with decreased IL-12p70 and IL-23 production. By intracytoplasmic staining, we confirmed that production of IL-12/23p40 by conventional dendritic cells (DCs) upon TLR ligation was decreased in frail patients. However, proportion of DCs and monocytes subsets, phenotypic maturation and proximal signaling events were found to be comparable in frail and healthy old subjects. These results suggest the importance of age-associated clinical parameters and not age by itself in the alteration of innate immune responses in old individuals and emphasis the importance of innate immune responses in the susceptibility of frail geriatric patients to infections.

A new device to mimic intermittent hypoxia in mice.

Intermittent hypoxia (hypoxia-reoxygenation) is often associated with cardiovascular morbidity and mortality. We describe a new device which can be used to submit cohorts of mice to controlled and standardised hypoxia-normoxia cycles at an individual level. Mice were placed in individual compartments to which similar gas flow parameters were provided using an open loop strategy. Evaluations made using computational fluid dynamics were confirmed by studying changes in haemoglobin oxygen saturation in vivo. We also modified the parameters of the system and demonstrated its ability to generate different severities of cyclic hypoxemia very precisely, even with very high frequency cycles of hypoxia-reoxygenation. The importance of the parameters on reoxygenation was shown. This device will allow investigators to assess the effects of hypoxia-reoxygenation on different pathological conditions, such as obstructive sleep apnoea or chronic obstructive pulmonary disease.

Design, synthesis, and structure-activity relationship studies of novel 3-alkylindole derivatives as selective and highly potent myeloperoxidase inhibitors.

Due to its production of potent antimicrobial oxidants including hypochlorous acid, human myeloperoxidase (MPO) plays a critical role in innate immunity and inflammatory diseases. Thus MPO is an attractive target in drug design. (Aminoalkyl)fluoroindole derivatives were detected to be very potent MPO inhibitors; however, they also promote inhibition of the serotonin reuptake transporter (SERT) at the same concentration range. Via structure-based drug design, a new series of MPO inhibitors derived from 3-alkylindole were synthesized and their effects were assessed on MPO-mediated taurine chlorination and low-density lipoprotein oxidation as well as on inhibition of SERT. The fluoroindole compound with three carbons in the side chain and one amide group exhibited a selectivity index of 35 (Ki/IC50) with high inhibition of MPO activity (IC50 = 18 nM), whereas its effect on SERT was in the micromolar range. Structure-function relationships, mechanism of action, and safety of the molecule are discussed.