The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-alpha-induced priming of the NADPH oxidase in human neutrophils.

Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α-induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α-induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

Design, characterization, and evaluation of peptide arrays allowing the direct monitoring of MMP activities.

Characterization of matrix metalloprotease (MMP) activities is of increasing interest for cancer prognosis or treatment follow-up. Indeed, MMP-1, -2 and -9 are widely involved in the growth of many tumors and progression steps such as angiogenesis, invasion, and metastasis. Fluorogenic peptide MMP substrates were previously synthesized with the aim of detecting MMP activities. One of their drawbacks is their limited solubility in biological media. Grafting them onto a solid support represented a novel way to yield efficient analysis devices whilst at the same time decreasing the quantities of peptides used. Novel peptide arrays were designed in order to detect MMP activities in biological fluids. Silicon plates were used as the solid support for the design of these novel tools. These were functionalized by organic self-assembled monolayers (SAMs) on which fluorogenic peptides were covalently coupled. SAM and peptide grafting on silicon plates were confirmed by epifluorescence, ellipsometry, and FT-IR analysis. Enzymatic assays were monitored by fluorescence spectrometry and showed that immobilized linear peptides were recognized and cleaved by MMPs.

Attenuated store-operated divalent cation entry and association between STIM1, Orai1, hTRPC1 and hTRPC6 in platelets from type 2 diabetic patients.

Agonist-evoked Ca(2+) entry has been reported to be enhanced in platelets from type 2 diabetic patients, which results in altered platelet responsiveness and cardiovascular complications. The present study is aimed to investigate whether store-operated divalent cation entry, a major Ca(2+) entry pathway, is altered in platelets from diabetic patients. Store-operated divalent cation entry was estimated by determination of Mn(2+) entry. Association between STIM1, Orai1, hTRPC1 and hTRPC6 was detected by co-immunoprecipitation and Western blotting. In the presence of specific purinergic and serotoninergic receptor antagonists Mn(2+) entry, induced by thapsigargin (TG), was reduced in platelets from diabetic donors as compared to healthy controls. Treatment with TG or the agonist thrombin enhanced co-immunoprecipitation of STIM1 with Orai1, hTRPC1 and hTRPC6 in platelets from healthy donors, a response that was significantly reduced in platelets from diabetic patients. Our results indicate that store-operated divalent cation entry is reduced in platelets from type 2 diabetic subjects, which is likely mediated by impairment of the association of STIM1 with the channel subunits Orai1, hTRPC1 and hTRPC6 and might be involved in the pathogenesis of the altered platelet responsiveness observed in diabetic patients.

Ca2+ leakage rate from agonist-sensitive intracellular pools is altered in platelets from patients with type 2 diabetes.

Platelets from patients with type 2 diabetes show abnormalities in intracellular Ca(2+) homeostasis that are involved in platelet hyperaggregability and the development of thrombotic complications. Different Ca(2+) transport mechanisms have been reported to be altered in platelets from patients with type 2 diabetes, including the sarcoendoplasmic and plasma membrane Ca(2+)-ATPases, plasma membrane Ca(2+) channels, or the Na(+)/Ca(2+) exchanger. Here, we have investigated whether passive Ca(2+) leak from the stores is altered in platelets from patients with type 2 diabetes. Resting cytosolic Ca(2+) concentration ([Ca(2+)](i)) was found to be greater in platelets from patients with type 2 diabetes than in healthy controls. In a Ca(2+)-free medium, platelet stimulation with thrombin or ADP evokes a rapid and transient increase in [Ca(2+)](i) that was found to be greater in patients with diabetes than in healthy controls. Sequential or combined inhibition of Ca(2+) extrusion and Ca(2+) sequestration into the stores reduced the difference between the responses to agonists in patients with diabetes and healthy controls, although agonist-induced Ca(2+) efflux from the stores was still significantly greater in patients with diabetes. Ca(2+) leak from the dense tubular system or the acidic stores, induced by a low concentration of thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ), respectively, was clearly greater in patients with diabetes than in controls, and was not significantly modified by treatment with 2-APB. These findings indicate that passive Ca(2+) leakage rate from the intracellular stores in platelets is significantly enhanced in patients with type 2 diabetes mellitus and this might explain the increased resting [Ca(2+)](i).

Novel cyclopeptides for the design of MMP directed delivery devices: a novel smart delivery paradigm.

PURPOSE: Matrix metalloproteinases (MMP) are a family of proteolytic enzymes, the expression of which in a key step of tumor progression has been better defined recently. The studies highlighted the ongoing need for very specific inhibitors, substrates or release devices designed to be selective for one or at least very few MMPs. METHODS: This report deals with the design, synthesis and in vitro evaluation of linear and especially novel cyclic peptidic moieties, embodying MMP cleavable sequences designed to answer these questions. FRET (fluorescence resonance energy transfer) labelling via chromophore-modified amino-acids was used to give access to enzyme kinetics. RESULTS: Evaluation of these peptides showed that cyclisation gives rise to high specificity for certain MMP, suggesting that this approach could provide very specific MMP substrate. Moreover, cyclic structures present a very good plasma stability. CONCLUSIONS: These original derivatives could allow the design of MMP-controlled delivery devices, the specificity of which will be retained in complex biological media and in vivo.

Biological analysis of endocrine-disrupting compounds in Tunisian sewage treatment plants.

Endocrin-disrupting compounds (EDCs) are frequently found in wastewater treatment plants (WWTPs). So far, research has been mainly focused on the detection of estrogenic compounds and very little work has been carried out on other receptors activators. In this study, we used reporter cell lines, which allow detecting the activity of estrogen (ERalpha), androgen (AR), pregnane X (PXR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and aryl hydrocarbon (AhR) receptors, to characterise the endocrine-disrupting profile of the aqueous, suspended particulate matter, and sludge fractions from three Tunisian WWTPs. The aqueous fraction exhibited estrogenic and androgenic activities. Suspended particulate matter and sludge extracts showed estrogenic, aryl hydrocarbon and pregnane X receptor activities. No GR, MR, or PR (ant) agonistic activity was detected in the samples, suggesting that environmental compounds present in sewage might have a limited spectrum of activity. By performing competition experiments with recombinant ERalpha, we demonstrated that the estrogenic activity detected in the aqueous fraction was due to EDCs with a strong affinity for ERalpha. Conversely, in the sludge fraction, it was linked to the presence of EDCs with weak affinity. Moreover, by using different incubation times, we determined that the EDCs present in suspended particulate matter and sludge, which can activate AhR, are metabolically labile compounds. Finally, we showed in this study that environmental compounds are mainly ER, AR, PXR, and AhR activators. Concerning AR and PXR ligands, we do not to know the nature of the molecules. Concerning ER and AhR compounds, competition experiments with recombinant receptor and analysis at different times of exposure of the AhR activation gave some indications of the compound's nature that need to be confirmed by chemical analysis.

Olive tree wood phenolic compounds with human platelet antiaggregant properties.

Oleuropein and (+)-cycloolivil are natural polyphenolic compounds with a significant radical scavenging activity present in olive tree. We have investigated the antiaggregant effects of oleuropein and (+)-cycloolivil isolated from an ethyl acetate extract of olive tree wood. Oleuropein and (+)-cycloolivil reduced the ability of thrombin to stimulate platelet aggregation. Both compounds reduced thrombin-evoked Ca(2+) release and entry to a similar extent to hydroxytyrosol. This effect was greater in platelets from patients with type 2 diabetes mellitus than in controls. Thrombin-, thapsigargin- and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-evoked protein tyrosine phosphorylation, which is involved in Ca(2+) signalling and platelet aggregation, is inhibited by oleuropein and (+)-cycloolivil. oleuropein and (+)-cycloolivil are natural oxygen radical scavengers that reduce thrombin-induced protein tyrosine phosphorylation, Ca(2+) signalling and platelet aggregation. These observations suggest that oleuropein and (+)-cycloolivil may prevent thrombotic complications associated to platelet hyperaggregability and be the base for the development of antiaggregant therapeutic strategies.

Enhanced expression of STIM1/Orai1 and TRPC3 in platelets from patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (DM2) is a metabolic syndrome that contributes to both macrovascular and microvascular disorders, where platelet hyperaggregability, associated to abnormal intracellular Ca(2+) homeostasis, plays an important role. We have now investigated the expression of different proteins associated to Ca(2+) entry, a major Ca(2+) signalling event. DM2 donors were randomly selected from normotensive patients with glycosylated Hb levels (HbA1c) over 6%. Control subjects were normal age- and gender-matched healthy people with HbA1c levels in the normal range (3.5-5%). Expression of TRPC1, 3 and 6, STIM1 and Orai1 was analyzed by Western blotting in DM2 patients and controls. Expression of TRPC1 in platelets from DM2 donors and controls was similar; however, expression of TRPC6 is reduced in platelets from DM2 patients as compared to healthy controls. We have found that expression of TRPC3, Orai1 and STIM1 is enhanced in DM2 subjects as compared to controls. Our findings provide an explanation to the enhanced Ca(2+) entry induced by physiological agonists in platelets from DM2 patients.

[Importance of HLA-G in therapy of cancer]. / Importance de HLA-G dans les stratégies thérapeutiques anti-cancéreuses.

HLA-G (Human Leucocyte Antigen-G) is a non-classical HLA class I molecule observed for the first time in human cytotrophoblast. Numbers of investigations have demonstrated that HLA-G was broader than originally thought. In fact, it is expressed in pathological contexts as well as in physiological contexts. This expression of HLA-G and its receptors in immunity cells confer to it a major role in immune responses. Good issues were described in organ transplantation when HLA-G was expressed. But, HLA-G transcripts and/or proteins expression in tumor tissues was associated with tumor genesis and cancer progression. A focus on the expression and the role of HLA-G in tumor context will be developed in this review. In addition, regulation of HLA-G will be treated to improve strategies of cancer therapy.

Characterization of the intracellular mechanisms involved in the antiaggregant properties of cinnamtannin B-1 from bay wood in human platelets.

Cinnamtannin B-1, a natural A-type proanthocyanidin recently identified as a radical scavenger component of Laurus nobilis L., exerts antiaggregant and antiapoptotic effects in human platelets. Here, we have investigated the intracellular mechanisms involved in the antiaggregant effects of cinnamtannin B-1. Cinnamtannin B-1 showed a greater free radical scavenging activity than vitamin C, vitamin E, or Trolox, among other antioxidants and reduced thrombin-evoked tubulin reorganization and platelet aggregation. Thrombin-evoked activation of Btk and pp60(src) was also inhibited by cinnamtannin B-1. In conclusion, we show that cinnamtannin B-1 is a powerful oxygen radical scavenger that reduces thrombin-evoked microtubular remodeling and activation of the tyrosine kinases Btk and pp60(src), which leads to inhibition of platelet aggregation. These observations suggest that cinnamtannin B-1 may prevent thrombotic complications associated to platelet hyperaggregability and hyperactivity, although further studies are necessary to establish appropriate therapeutic strategies.

Caspases 3 and 9 are translocated to the cytoskeleton and activated by thrombin in human platelets. Evidence for the involvement of PKC and the actin filament polymerization.

Platelets express, among others, initiator caspase 9 and effector caspase 3. Upon activation by physiological agonists, calcium ionophores or under shear stress they might develop apoptotic events. Although it is well known that the cytoskeletal network plays a crucial role in apoptosis, the relationship between caspases 3 and 9 and the cytoskeleton is poorly understood. Here we demonstrate that the physiological agonist thrombin is able to induce activation of caspases 3 and 9 in human platelets and significantly increases the amount in the cytoskeleton of the active forms of both caspases and the procaspases 3 and 9. After stimulation with thrombin the amount of active caspases 3 and 9 in the cytosolic and cytoskeletal fractions were significantly reduced in Ro-31-8220-treated cells, which demonstrates that caspases activation and association with the cytoskeleton needs the contribution of PKC. Inhibition of actin polymerization by cytochalasin D inhibits translocation and activation of both caspases, suggesting that thrombin stimulates caspase 3 and 9 activation and association with the reorganizing actin cytoskeleton. Finally, our results show that inhibition of thrombin-induced caspase activation has no effect on their translocation to the cytoskeleton although impairment of thrombin-evoked caspase translocation has negative effects on caspase activity, suggesting that translocation to the cytoskeleton might be important for caspase activation by thrombin in human platelets.

Estrogens and antiestrogens activate hPXR.

The pregnane X receptor (PXR, NR1I2) and the estrogen receptors (ERalpha, NR3A1 and ERbeta, NR3A2) bind a large number of compounds, including environmental pollutants and drugs, which exhibit remarkably diverse structural features. This prompted us to investigate if ER ligands could be PXR activators. We focused our attention on known estrogens from various chemical classes: physiological and synthetic estrogens and antiestrogens, plant and fungus estrogens, and other man-made chemicals belonging to phthalate plasticizers, surfactant-derived alkylphenols and cosmetics. Altogether, nearly 50 compounds were thus analyzed for their ability to activate human PXR in stably transfected cells, HGPXR cells, derived from HeLa cells and expressing luciferase under the control of a chimeric hPXR. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 and 2B6 expressions in a primary culture of human hepatocytes. A significant proportion (54%) of compounds with estrogenic activity or able to bind ER were found to be hPXR activators: in particular, antiestrogens, mycoestrogens and phthalates. An even greater proportion is observed if estrogenic pesticides are included. Altogether, these results raise the question of the meaning and consequences of compounds with double PXR/ER activation ability.

[Endocrine xenoestrogenics disrupters: molecular mechanisms and detection methods]. / Les perturbateurs endocriniens xénooestrogéniques: mécanismes moléculaires et méthodes de détection.

The attention paid to endocriniens modulators for purpose micropolluants (endocrine disrupters) has been increasingly studied these last years particularly on animals. The results of this study raised big concerns from Doctors and Biologists on the eventual risks human health can face. Indeed, endocrine systems of the body play an essential and pervasive role in both the short- and long-term regulation of metabolic processes. Nutritional, behavioural, and reproductive processes are intricately regulated by endocrine systems, as are growth (including bone growth/remodelling), gut, cardiovascular, and kidney function and responses to all forms of stress. Disorders of any of the endocrine system, involving both over- and under-active hormone secretion, result inevitably in disease, the effects of which may extend to many different organs and functions and are often debilitating or life-threatening. Viewed from this general perspective, the threat posed from environmental chemicals with endocrine activity (either agonist or antagonistic) is potentially serious. However, the fact that humans and wildlife are exposed to such chemicals does not necessarily mean that clinically manifest disturbance of the relevant endocrine system will result, because much depends on the level and duration of exposure and on the timing of exposure. Indeed, a large numbers of environmental estrogens are suspected of altering human health as well as the marine ecosystem balance. The objective of this review is to study the different molecular mechanisms of these xenoestrogenes micropolluants, in order to emphasize their potential risk and to present some of the different experimental methods for their detection.

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets.

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.

Expression of uPA, tPA, and PAI-1 in Calcified Aortic Valves.

Purpose. Our physiopathological assumption is that u-PA, t-PA, and PAI-1 are released by calcified aortic valves and play a role in the calcification of these valves. Methods. Sixty-five calcified aortic valves were collected from patients suffering from aortic stenosis. Each valve was incubated for 24 hours in culture medium. The supernatants were used to measure u-PA, t-PA, and PAI-1 concentrations; the valve calcification was evaluated using biphotonic absorptiometry. Results. Aortic stenosis valves expressed normal plasminogen activators concentrations and overexpressed PAI-1 (u-PA, t-PA, and PAI-1 mean concentrations were, resp., 1.69 ng/mL ± 0.80, 2.76 ng/mL ± 1.33, and 53.27 ng/mL ± 36.39). There was no correlation between u-PA and PAI-1 (r = 0.3) but t-PA and PAI-1 were strongly correlated with each other (r = 0.6). Overexpression of PAI-1 was proportional to the calcium content of the AS valves. Conclusions. Our results demonstrate a consistent increase of PAI-1 proportional to the calcification. The overexpression of PAI-1 may be useful as a predictive indicator in patients with aortic stenosis.

Golimumab and malignancies: true or false association?

Malignancy is one of the comorbidities linked to golimumab, a biological TNF-α blocker. In this systematic review and meta-analysis, we searched different databases and analyzed original publications to elucidate the remaining open question about the real association of malignancies with golimumab therapy. The most frequent cancer in patients treated with golimumab, in association or not with methotrexate, is the lung adenocarcinoma. However, lymphoma is not very commonly represented in these patients. We show that there is no major and evident risk of malignancies associated with golimumab in current scientific literature. An increased risk of malignancies may be associated with golimumab, but this warrants further clinical confirmation. Also, this risk mentioned in different studies must be taken with caution because of number of limits and biases.

Effect of endocrine disruptor pesticides: a review.

Endocrine disrupting chemicals (EDC) are compounds that alter the normal functioning of the endocrine system of both wildlife and humans. A huge number of chemicals have been identified as endocrine disruptors, among them several pesticides. Pesticides are used to kill unwanted organisms in crops, public areas, homes and gardens, and parasites in medicine. Human are exposed to pesticides due to their occupations or through dietary and environmental exposure (water, soil, air). For several years, there have been enquiries about the impact of environmental factors on the occurrence of human pathologies. This paper reviews the current knowledge of the potential impacts of endocrine disruptor pesticides on human health.

TNF-alpha and its inhibitors in cancer.

Tumor necrosis factor (TNF)-alpha is implicated in the same time in apoptosis and in cell proliferation. TNF-alpha not only acts as pro-inflammatory cytokine conducing to wide spectrum of human diseases including inflammatory diseases, but can also induce tumor development. The molecular mechanisms of TNF-alpha functions have been intensively investigated. In this review we covered TNF-alpha, the molecule, its signaling pathway, and its therapeutic functions. We provide a particular insight in its paradoxical role in tumor promotion and in its use as anti-tumor agent. This review considers also the recent findings regarding TNF-alpha inhibitors, their pharmacokinetics, and their pharmacodynamics. Six TNF-alpha inhibitors have been considered here: Infliximab, Adalimumab, Golimumab, CDP870, CDP571, Etanercept, and Thalidomide. We discussed the clinical relevance of their functions in treatment of several diseases such as advanced inflammatory rheumatic and bowel disease, with a focus in cancer treatment. Targeting TNF-alpha by these drugs has many side effects like malignancies development, and the long-term sequels are not very well explored. Their efficacy and their safety were discussed, underscoring the necessity of close patients monitoring and of their caution use.

Homocysteine induces caspase activation by endoplasmic reticulum stress in platelets from type 2 diabetics and healthy donors.

Diabetes mellitus is a disease characterised by hyperglycaemia and associated with several cardiovascular disorders, including angiopathy and platelet hyperactivity, which are major causes of morbidity and mortality in type 2 diabetes mellitus. In type 2 diabetic patients, homocysteine levels are significantly increased compared with healthy subjects. Hyperhomocysteinaemia is an independent risk factor for macro- and microangiopathy and mortality. The present study is aimed to investigate the effect of homocysteine on platelet apoptosis. Changes in cytosolic or intraluminal free Ca(2+) concentration were determined by fluorimetry. Caspase activity and phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha) were explored by Western blot. Our results indicate that homocysteine releases Ca(2+) from agonist sensitive stores, enhances eIF2alpha phosphorylation at Ser(51) and activates caspase-3 and -9 independently of extracellular Ca(2+). Homocysteine induced activation of caspase-3 and -9 was abolished by salubrinal, an agent that prevents endoplasmic reticulum (ER) stress-induced apoptosis. Homocysteine-induced platelet effects were significantly greater in type 2 diabetics than in healthy subjects. These findings demonstrate that homocysteine induces ER stress-mediated apoptosis in human platelets, an event that is enhanced in type 2 diabetic patients, which might be involved in the pathogenesis of cardiovascular complications associated with type 2 diabetes mellitus.

Acidic-store depletion is required for human platelet aggregation.

Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets.