Methionine-Enkephalin Primes Human Neutrophils for Enhanced Superoxide Anion Production

Methionine-enkephalin (MENK) is not an effective stimulus for inducing the superoxide (O^-₂) generation of human neutrophils, but it enhanced the O^-₂ generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or human recombinant interferon gamma (hrIFNγ) when the cells had been preincubated with MENK for 30 min at 37°C. The priming effect of MENK was not observed with stimulus such as lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA). The enhancing effect of MENK was abrogated if cells were treated with protein kinase C (PKC) inhibitor H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before fMLP or IFNγ. This finding indicates that MENK is a potent modulator of human neutrophils and can contribute to inflammatory process.     

Protein-A activates membrane bound multicomponent enzyme complex, NADPH oxidase in human neutrophils

Protein-A, 42KD cell wall glycoprotein of S. aureus Cowan I enhance mononuclear and polymorphonuclear cell counts in vivo and possesses antitoxic, antitumor, properties. In order to explain the mechanism of its function, the respiratory burst phenomenon in cell and cell free system was studied using lucigenin-dependent chemiluminescence technique. A dose dependent increase in protein A-mediated generation of superoxide radical was observed in resting and PMA stimulated neutrophils. Superoxide dismutase (SOD) was used to confirm the production of superoxide radicals (O^-₂). To understand the mechanism of protein-A induced O^-₂ generation; NADPH oxidase activity was measured in cell free system using NADPH as a substrate. A significant increase in NADPH oxidase activity was observed in the membrane and post-nuclear supernatant fraction of activated human neutrophils. Cytosolic fraction showed slight enzyme activation. Protein A (SpA)-induced NADPH oxidase activation in the membrane fraction was observed even in the absence of the substrate NADPH. These data indicate that protein A attenuate the NADPH oxidase system to produce O^-₂ radicals.     

芒柄花黄素对超氧阴离子及羟自由基清除作用的研究

目的:探讨芒柄花黄素体外对氧自由基的抑制和清除作用。方法:采用分光光度法检测芒柄花黄素对超氧阴离子自由基(O2-)和羟自由基(.OH)的清除及抑制作用。结果:芒柄花黄素终浓度为10、20、40μg.ml-1时,在体外对O2-的清除率分别为42.19%,56.64%及65.43%,对O2-生成的抑制率分别为31.06%,44.72%及63.35%;而对.OH的清除率分别为34.98%,41.06%及61.60%。结论:芒柄花黄素对O2-具有明显的抑制及清除作用,其对.OH也有明显的清除作用。     

Modulation of Cytoskeleton Assembly Capacity and Oxidative Response in Aged Neutrophils

Several reports have emphasized that aged polymorphonuclear cells (PMN) exhibit an impairment of superoxide anion (O^-₂) generation when triggered with formyl-methionyl-leucine-phenylalanine (FMLP) in comparison to the younger counterpart. Since microfilaments and microtubules are involved in PMN-mediated functions, in a group of old donors we assessed the effects of either actin stabilizing and disrupting agents, i.e. phalloidin and cytochalasin B, or microtubule stabilization or disruption by taxol and colchicine, respectively, on FMLP-triggered neutrophil oxidative responsiveness. Results show that phalloidin treatment, at a concentration ranging from 10^-6 to 10^-8 M, gave rise to an inhibition of O^-₂ release by aged PMN, while the same effect was seen in similarly treated young cells at a concentration of 10^-7 M only. On the contrary, cytochalasin B pretreatment led to an enhancement of O^-₂ generation in both young and aged neutrophils, even if to a lower extent in the latter group. At the same time, taxol at 10^-8 M strength inhibited young cell responsiveness, while no effects were induced by colchicine treatment. Quite interestingly, elderly neutrophil function was negatively modulated by both microtubule affecting compounds.

Alltogether, these findings suggest the possible relevance of cytoskeletal affecting compounds in the modulation of FMLP-stimulated O^-₂ release during senescence.     

Adiponectin inhibits superoxide generation by human neutrophils

Adiponectin (Ad), a member of the adipocytokine family, has been reported to possess antiinflammatory properties. We investigated the effects of full-length human Ad (hAd) on phorbol 12-myristate 13-acetate (PMA)-induced O2-* generation by human neutrophils. hAd, even at the lowest tested concentration of 0.001 microg/ml, after 30-min pretreatment of cells, significantly inhibited O2-* generation by neutrophils stimulated with PMA (100 nM). However, no relation between the dose of hAd and extent of inhibition of PMA-induced O2-* generation was observed with increasing the concentration of hAd up to 1 microg/ml. hAd also significantly inhibited neutrophil O2-* generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (100 microM) and diacylglycerol (500 nM), as well as the PMA-induced neutrophil nitroblue tetrazolium reduction and H2O2 formation. Pretreatment of neutrophils with pronase-digested hAd failed to inhibit the PMA-induced O2-* generation. For the first time, this study revealed that Ad inhibited O2-* generation by neutrophils, possibly through regulation of NADPH oxidase.     

Anti-inflammatory effect of lemon mucilage: in vivo and in vitro studies

The mucilage extracted from a lemon juice centrifugation pulp was studied for its anti-inflammatory effect in rat. In vivo the lemon mucilage significantly inhibited carrageenan-induced edema in rat paw from 59% to 73.5% showing the highest effect at the third hour. In vitro, at the doses of 10^-8, 10^-6, 10^-4 or 10^-2 mg/mL the lemon mucilage stimulated the superoxide anion production in rat testing neutrophils in whole blood but inhibited it in FMLP stimulated cells at the dose of 10^-2 mg/mL. The neutrophils of rats receiving p.o. the lemon mucilage for 21 days showed a significant decrease of 45.5% in O₂^- generation after FMLP stimulation, and a not-significant increase after phorbol-12-myristate-13-acetate (PMA) or zymosan stimulation. Since the activity on zymosan- and PMA-induced O₂^- production was not significant, the inhibition exerted by FMLP in rat neutrophils occurred mainly through the blockade of phospholipase D.     

Dipalmitoylphosphatidylcholine modulates inflammatory functions of monocytic cells independently of mitogen activated protein kinases

Phosphatidylcholine (PC) is the major phospholipid of pulmonary surfactant and it is hypothesized that PC and its subspecies modulate the functions of alveolar macrophages. The most abundant of these subspecies is dipalmitoylphosphatidylcholine (DPPC). This study was undertaken to determine the effect of PC on monocyte function using a human monocytic cell line, MonoMac-6 (MM6). This study showed that preincubation of MM6 cells with DPPC at 125 microg/ml for 2 h inhibited the oxidative response to either zymosan or phorbol-12-myristate-13-acetate (PMA) by 30% (P < 0.001). This inhibition with DPPC was independent of LPS priming. When DPPC was replaced with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) there was no inhibition and in contrast a significant increase in oxidant production was observed. We also demonstrated that total PC (tPC; a heterogeneous species of PC from egg) and DPPC but not PAPC significantly inhibited the release of TNF-alpha from MM6 cells (P < 0.05). DPPC did not inhibit phosphorylation of the mitogen activated protein kinases (MAPKs) p44/p42 or p38 in stimulated cells. Measurements of membrane fluidity with spin label EPR spectroscopy indicate that DPPC incorporation significantly alters the membrane fluidity of MM6 cells. These results suggest that DPPC, the major component of pulmonary surfactant, may play a role in modulating leucocyte inflammatory responses in the lung. This may in part be related to membrane effects but does not include alterations in p44/p42 or p38 MAPK signalling.     

Superoxide generation from human polymorphonuclear leukocytes by liposome-encapsulated hemoglobin

We investigated the interactions between liposome-encapsulated hemoglobin (Neo Red Cells: NRC) and human polymorphonuclear leukocytes as assessed by superoxide generation. NRC triggered superoxide generation from neutrophils in a dose-dependent manner. Empty liposomes also induced superoxide production of neutrophils. Superoxide generation of neutrophils induced by phorbol myristate acetate (PMA) was delayed but intensified both by NRC and empty liposomes. The intensity of superoxide generation induced by NRC was smaller than that by the empty liposomes. As NRC contained superoxide dismutase (SOD) that was copurified with hemoglobin from red blood cells and its activity remained, SOD contained in NRC may partially eliminate superoxide.     

Use of Liposomes as an Immunopotentiating Delivery System: in Perspective of Vaccine Development

Liposomes have been widely used to deliver antigens to the antigen-presenting cells (APCs) and also to modify their immunological behaviour in model animals. We recently demonstrated the potential of yeast lipid liposomes to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells. Interestingly, studies in the present report revealed that antigen encapsulated in yeast lipid liposomes could be successfully delivered simultaneously into the cytosolic as well as endosomal processing pathways of APCs, leading to the generation of both CD4+ T helper and CD8+ cytotoxic T cells. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (PC) liposomes, just like its free form, has inefficient access to the cytosolic pathway of major histocompatibility complex (MHC) I dependent antigen presentation and failed to generate antigen specific CD8+ cytotoxic T-cell response. However, both egg PC as well as yeast lipid liposomes have elicited strong antigen specific antibody responses in immunized animals. These results imply usage of liposome encapsulated antigen as potential candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses.     

Induction of Autoimmune Prostatitis Using Liposomes Is Associated to Peritoneal Cells Activation

PROBLEM: Study and characterization of rat peritoneal cells (PC) involved in the induction of autoimmune prostatitis after the intraperitoneal administration of native extract of accessory glands (RAG) associated with liposomes (RAGL). METHOD OF STUDY: Induction of the autoimmune response in normal recipients by transferring PC or adherent-PC loaded with RAGL (RAGL-PC), but not with PC loaded with empty liposomes (L-PC). Characterization of the morphology, the ultrastructure, and the phenotype of L-PC or RAGL-PC. Study of the respiratory burst by the nitroblue tetrazolium (NBT) reduction assay after stimulation with phorbol myristate acetate (PMA) in both L-PC and RAGL-PC. RESULTS: Liposomes attached to the cell surface of the Mφ were observed by electron microscopy. FACS analyses showed a similar staining pattern with high expression of Ia molecules on L-PC and RAGL-PC compared with controls. PMA-stimulated L-PC or RAGL-PC markedly reduced the NBT compared with controls. CONCLUSION: Our results suggest that the effective uptake of liposomes and the initial activation of PC together with a prolonged stimulatory effect help to disrupt the tolerance state. The present experimental model is an interesting approach to further characterize events associated with antigenic presentation when an autoimmune response is triggered.     

Phorbol myristate acetate-induced neutrophil autotoxicity

We have previously shown that in the presence of phorbol myristate acetate (PMA), neutrophils kill neoplastic cells as well as themselves. This PMA-induced neutrophil autotoxicity was markedly inhibited by catalase, suggesting that H₂O₂ directly or indirectly played an important role. In this study we compared PMA and H₂O₂ toxicity against human neutrophils. The effect of H₂O₂ was faster and more sensitive to catalase and serum than that of PMA. Sodium azide markedly enhanced the effect of H₂O₂ but not that of PMA. In contrast, methionine and histidine prevented the toxicity of PMA, while they had no effect on H₂O₂ toxicity. The results suggest that PMA-induced neutrophil autotoxicity is not mediated by H₂O₂ alone.     

Cristallographic behaviour of fluoridated hydroxyapatites containing Mg and CO3 ions

Fluoridated hydroxyapatites containing small amounts of magnesium and carbonate ions were synthesized at 80 and 60° C to examine their inhibiting properties regarding apatite crystal growth, in contrast to the promoting action of fluoride. The shortening of a-axis and c-axis dimensions of the apatite crystals, as revealed by X-ray diffraction analysis, suggested that both magnesium and carbonate ions were substituted into the apatite crystals. The a-axis dimensions also decreased with the degree of fluoridation. The infrared spectra due to CO²⁻₃ ions at 875 cm⁻¹ were shifted with increasing fluoride content. The overall crystallinity was inhibited in comparison with that of Mg and CO₃-free fluoridated hydroxyapatites, but recovered considerably with increased fluoride content. The apparent solubility of the apatites at pH 4.0 and 37°C was higher than that of Mg and CO₃-free fluoridated hydroxyapatites at lower fluoride contents, but gradually approached the latter at higher fluoride content. After 1 month's incubation, dicalcium phosphate dihydrate was formed from fluoride-free Mg-CO₃ apatite synthesized at 60°C.     

Effects of estrogen on female mouse thymus, with special reference to ER-mRNA and T cell subpopulations

The effect of estrogen (E), a female sex steroid, on the thymus tissues from castrated female mice treated with E was examined by molecular biologic, microscopic and flowcytometric techniques.

First, using an oligoprobe for E receptor (ER)-messenger RNA (ER-mRNA), one hybridized band was found at 6.2 kilobase (kb) in mouse thymus tissue, as was also the case in the human breast cancer MCF-7 cell line. ER-mRNA level in the E-treated animals was almost 3 times that in oil-treated controls.

Secondly, an electron microscopic observation indicated E treatment to bring about apoptosis of thymocytes (T cell) which were embraced by thymic stromal cells (possibly phagocytic in nature) and/or ballooning of the endoplasmic reticulum in the epithelial cells with abundant lipid droplets.

Thirdly, flow cytometric analysis demonstrated E to induce the change of T cell subpopulations: an increase in helper/inducer (L₃T⁺₄Lyt⁻₂) cells with decrease in the double positive (L₃T⁺₄Lyt⁺₂) cells.

It follows from the above findings that E may cause morphologic changes in the thymus closely related to T cell differentiation. In addition, these changes appear to derive mainly from E-induced tissue-specific gene expression including that of ER-mRNA.     

Liposomes as carriers for allergy immunotherapy

With the aim of obtaining an efficient but safer vaccine for allergy immunotherapy, the possibility of using liposomes as adjuvants has been considered given their proven low toxicity, adjuvant properties and ability to maintain the encapsulated substance in their interior for some time in vivo. Different methods of encapsulating allergenic extracts into neutral, positively, and negatively charged DPPC: cholesterol liposomes have been investigated and the immune response provoked by these in mice was studied and compared to the immune response to free allergen or other adjuvants such as aluminium hydroxide. The results obtained show that allergen encapsulated in all three types of liposomes elicit an increase in specific IgG levels higher than that provoked by free allergen, however, both encapsulation efficiency and specific IgG titre were higher when positively charged (DPPC: cholesterol stearylamine) liposomes were used. Specific IgE levels to allergen in positive and neutral liposomes was lower than to allergen in negative liposomes or adsorbed to Al(OH)3. No difference were found in the behaviour of liposomes prepared by different methods (the results were obtained from pooled sera from different groups of mice so there is no statistical data). These results confirm the immunoadjuvant effect of liposomes for allergy immunotherapy. Further studies to determine their lack of toxicity, pharmacokinetic studies and human clinical studies are necessary to confirm their adequacy for human use and advantage over current immunotherapy methods.     

Metalloproteases and Serineproteases are Involved in the Cleavage of the Two Tumour Necrosis Factor (TNF) Receptors to Soluble Forms in the Myeloid Cell Lines U-937 and THP-1

The proteolytic processing of the two TNF receptors (TNF-R55 and TNF-R75) into soluble forms was investigated in the myeloid cell lines U-937 and THP-1. Phorbol myristate acetate (PMA) rapidly stimulated release of soluble forms of both TNF-receptors. Incubations were made with PMA and protease inhibitors directed against different target protease groups. The serineprotease inhibitors TPCK and dichloroisocoumarin and the metalloprotease inhibitor 1, 10-phenanthroline reduced PMA-induced release of both soluble receptor forms with about 60–70%. Furthermore, 1, 10-phenanthroline also reduced PMA-induced down-regulation of TNF-receptors in both cell lines as judged by TNF-binding to cells. Reduced down-regulation and TNF-receptor shedding by 1, 10-phenanthroline was reversed by Zn²⁺, indicating involvement of a Zn²⁺-dependent metalloprotease. Thus, both serine proteases and metalloproteases are involved in the processing of TNF-receptors.     

Progestins used in hormonal replacement therapy display different effects in coronary arteries from New Zealand white rabbits

Objectives: The aim of this study was in an animal model to assess the vascular effects of different progestins commonly used in hormonal replacement treatment. Methods: Fifty-six non-atherosclerotic, ovariectomized New Zealand white rabbits were randomized into seven groups: (1) medroxyprogesterone acetate (MPA), (2) norethisterone acetate (NETA), (3) conjugated equine estrogens (CEE), (4) 17-β-estradiol (E2), (5) MPA+CEE, (6) NETA+E2, (7) or placebo (n=8) and given hormonal treatment through the diet for 4 weeks. Ring segments from the left proximal coronary artery and from the distal part of the left anterior descending coronary artery were microdissected and mounted for isometric tension recordings in a myograph. The vasoconstrictory responses induced by potassium, endothelin-1, calcium and N_w-nitro- -arginine methyl ester, and the vasodilatory response induced by acetylcholine and sodiumnitroprusside were investigated. The maximum contraction/relaxation (E_max) and the concentration required to induce half the maximum response (EC₅₀) were determined. EC₅₀ values were expressed as the negative logarithm to the molar concentration, pD₂=−log EC₅₀. Results: Treatment with MPA alone caused when compared to treatment with NETA an increase in tension development in the distal coronary artery after the addition of potassium (6.36±0.36 versus 4.31±0.42 P<0.005) (single dose response, mN/mm, mean±S.E.M.) and endothelin-1 (9.41±0.82 versus 6.43±0.73 P<0.05) (E_max, mN/mm, mean±S.E.M.). Treatment with MPA compared to placebo caused an endothelin-1 induced increase of E_max in the distal coronary artery (9.21±0.87 versus 6.51± 0.65 P<0.05) and a calcium induced increase of pD₂ in both coronary arteries (2.98±0.19 versus 2.42±0.12 P<0.05, proximal coronary artery) (3.26±0.09 versus 2.9±0.1 P<0.05, distal coronary artery) (pD₂, mean±S.E.M.). Treatment with NETA compared to placebo in the proximal coronary artery, after the addition of sodiumnitroprusside caused a decrease of pD₂ (5.33±0.19 versus 5.94±0.13 P<0.05). Treatment with E2 compared to treatment with CEE in the proximal coronary artery caused a decrease of pD₂ after the addition of sodiumnitroprusside (5.00±0.16 versus 5.77±0.28 P<0.05). No significant differences were found between MPA+CEE and NETA+E2. Conclusion: Treatment with MPA alone seems to enhance the contractile response to potassium and endothelin-1 in the distal coronary artery compared to NETA, indicating that different progestins used in hormonal replacement treatment may display different effects on contractile functions of coronary arteries.     

Endothelin-1 and NO2/NO3 Circulating Levels after Short-Term (1h) Oxygen Supplementation in Patients with Chronic Respiratory Failure During Long-Term Oxygen Treatment (LTOT)

The effect of acute oxygen administration on endothelin-1 (ET-1) and nitrates (NO^-₂/NO^-₃), the latter as stable end products of nitric oxide (NO), were evaluated in arterial and venous blood of chronic respiratory failure (CRF) patients underwent to a continuous long-term oxygen therapy (LTOT). After one hour of oxygen supplementation, ET-1 showed a marked and significant decrease more pronounced in venous blood whereas no statistical change in NO^-₂/NO^-₃ concentrations were observed in both arterial and venous blood. There are evidences for increased expression of ET-1 in several pulmonary diseases and for ET-1 plasma reduction in Adult Respiratory Distress Syndrome (ARDS) in patients which recovered. ET-1 is a potent human pulmonary vessel constrictor and may have other effects including plasma exudation, increased mucus secretion and a increased fibrinogenesis. Our data suggest that the improvement in air function, evaluated in part by the decreased release of inflammatory mediators and mainly by reduction in the pulmonary arterial resistence, may be a consequence of the decrease in ET-1 content in the lungs of CRF patients treated with LTOT.     

A rapid ELISA for screening aflatoxin B1 in animal feed and feed ingredients in Indonesia

To facilitate a sustainable aflatoxin management system in Indonesia, a simple, rapid and effective Enzyme Linked Immunosorbent Assay (ELISA) test for screening aflatoxin B₁ (AFB₁) in animal feed and feed ingredients was developed. Anti-AFB₁ polyclonal antibodies were produced against AFB₁-BSA and AFB₁-KLH immunogens. Using AFB₁ conjugated to horseradish peroxidase (HRP) as an enzyme marker in a direct competitive ELISA, an IC₅₀ of 0.85±0.15 µg/kg and a detection limit (IC₁₅) of 0.18±0.06 µg/kg of AFB₁ were achieved. The assay was highly specific to AFB₁ with very little cross reaction with other aflatoxin congeners (AFB₂ 0.9%, AFG₁ 3.1% and AFG₂ 1.2%) and metabolites. The ELISA was tolerant to methanol (up to 60%) and pH (pH 7.2-9.6) without significantly affecting the overall performance and was not affected by interferences from the animal feed and corn samples. Satisfactory recovery results were obtained from the spike and recovery study (77-97% recovery for 10-252 µg/kg). A pilot survey conducted on corn and animal feed samples collected from the local feed factories and poultry shops indicated that significant amounts of corn and animal feeds were contaminated by aflatoxin B₁ greater than the MRL (50 µg/kg).     

Comparison of the long-term effects of oral estriol with the effects of conjugated estrogen on serum lipid profile in early menopausal women

Objective: We investigated the long-term effects of oral estriol (E₃) on serum levels of total cholesterol (t-Cho), high-density lipoprotein cholesterol (HDL-Cho), low-density lipoprotein cholesterol (LDL-Cho), and triglycerides in early menopausal women. Methods: We studied 67 healthy early menopausal women who were treated for 48 months with 2.0 mg of E₃ plus 2.5 mg of medroxyprogesterone acetate daily (E₃ group, n=21), 0.625 mg of conjugated estrogen plus 2.5 mg of medroxyprogesterone acetate daily (CE group, n=19), or 1.0 μg of 1-hydroxyvitamin D₃ daily or 1.8 g of calcium lactate containing 250 mg of elemental calcium daily (control group, n=27). The serum levels of t-Cho, HDL-Cho, LDL-Cho, and triglycerides were evaluated at baseline and every 6 months. Results: After 48 months of treatment, the t-Cho decreased significantly by 4.3±2.1% (mean±SE) from baseline in the E₃ group, did not change in the CE group (−1.9±2.1%), and significantly increased (5.4±3.4%) in the control group. The HDL-Cho significantly increased in the CE group (10.7±2.4%), but not in the E₃ group (3.8±3.3%) or in the control group (−3.6±3.0%). The LDL-Cho significantly decreased in the CE group (−11.4±4.0%), did not change in the E₃ group (−5.2±3.6%), and significantly increased in the control group (11.8±6.3%). The triglyceride level decreased significantly in the E₃ group (−6.7±4.9%), whereas it significantly increased in the CE group (17.6±11.4%), and did not change in the control group (6.1±6.4%). Conclusions: Oral E₃ prevented a postmenopausal rise in the t-Cho. Oral estriol did not induce the hypertriglyceridemia that was seen after treatment with conjugated estrogen. Oral E₃ may be a useful alternative therapy in women with hypertriglyceridemia and in women who are reluctant to continue conventional hormone replacement therapy because of uterine bleeding.     

Release of Leukotrienes and Histamine by the Isolated Anaphylactic Heart

The purpose of this study was to demonstrate the ability of heart tissue to release the mediators of anaphylaxis after antigenic challenges. Guinea pigs were sensitized with ovalbumin. Hearts were exised, perfused in a langendorff apparatus, and challenged with a bolus injection of ovalbumin. Analysis of the perfusates demonstrated the presence of histamine as determined by radioenzymatic assay. Histamine release was observed to be maximum after 2 min (8±1 nmol) of perfusion, then decreased to baseline level. The heart also released LTB₄, LTC₄, LTD₄, and LTE₄ as determined by high performance liquid chromatography and bioassays. The release of LTC₄ occurred rapidly, reaching maximum after 2 min (4.2±1 pmol) and then returned to baseline level. Although the release of LTD₄ paralleled the release of LTC₄, it reached a maximum after 5 min (7.7±2 pmol). LTE₄ was detected after 10 min and was undetectable after 15 min. Maximum release of LTB₄ was observed after 5-10 min (15±3 pmol) and was no longer detectable after 15 min. These results indicate that the isolated sensitized heart undergoing antigenic challenge releases leukotrienes and histamine suggesting the cardiac anaphylaxis might occur by the locally released mediators.