Search

At inflammatory sites, cytotoxic agents are released and generated from invading immune cells and damaged tissue cells. The further fate of the inflammation highly depends on the presence of antagonizing principles that are able to inactivate these host-derived cytotoxic agents. As long as the affected tissues are well equipped with ready-to-use protective mechanisms, no damage by cytotoxic agents occurs and resolution of inflammation is initiated. However, long-lasting and severe immune responses can be associated with the decline, exhaustion, or inactivation of selected antagonizing principles. Hence, cytotoxic agents are only partially inactivated and contribute to damage of yet-unperturbed cells. Consequently, a chronic inflammatory process results. In this vicious circle of permanent cell destruction, not only novel cytotoxic elements but also novel alarmins and antigens are liberated from affected cells. In severe cases, very low protection leads to organ failure, sepsis, and septic shock. In this review, the major classes of host-derived cytotoxic agents (reactive species, oxidized heme proteins and free heme, transition metal ions, serine proteases, matrix metalloproteases, and pro-inflammatory peptides), their corresponding protective principles, and resulting implications on the pathogenesis of diseases are highlighted.

The heme protein myeloperoxidase (MPO) is a major constituent of neutrophils. As a key mediator of the innate immune system, neutrophils are rapidly recruited to inflammatory sites, where they recognize, phagocytose, and inactivate foreign microorganisms. In the newly formed phagosomes, MPO is involved in the creation and maintenance of an alkaline milieu, which is optimal in combatting microbes. Myeloperoxidase is also a key component in neutrophil extracellular traps. These helpful properties are contrasted by the release of MPO and other neutrophil constituents from necrotic cells or as a result of frustrated phagocytosis. Although MPO is inactivated by the plasma protein ceruloplasmin, it can interact with negatively charged components of serum and the extracellular matrix. In cardiovascular diseases and many other disease scenarios, active MPO and MPO-modified targets are present in atherosclerotic lesions and other disease-specific locations. This implies an involvement of neutrophils, MPO, and other neutrophil products in pathogenesis mechanisms. This review critically reflects on the beneficial and harmful functions of MPO against the background of immune response.

Objectives The enzyme lactoperoxidase (LPO), which is released into several body fluids like saliva, is an essential part to maintain the oral bacterial homeostasis by catalysing the oxidation of thiocyanate (SCN − ) to hypo-thiocyanite ( − OSCN). The formation of unreactive redox intermediates (like Compound II) leads to a decreased pseudo-halogenating enzyme activity, which is associated with a higher risk for oral infections. According to former studies with bovine LPO selected flavonoids were tested in respect to their potential to reactivate the enzymatic activity in a more physiological, human salivary system. Design Saliva samples from healthy donors were collected and characterized by using several gel staining methods and immunoblotting. Afterwards kinetic measurements were performed by applying the TNB-assay to evaluate the pseudo-halogenating salivary peroxidase (SAPX) activity. The measurements were performed in the presence of excess H 2 O 2 to simulate pro-inflammatory conditions. Moreover selected flavonoids or an ethanolic extract of Tormentillae rhizoma were applied to test their regenerating effect on the LPO-derived − OSCN production. Results Despite the complex protein composition of the collected saliva samples, an SAPX-derived pseudo-halogenating activity could be identified. The − OSCN regenerating effects of the tested polyphenols were completely comparable to previous in vitro experiments with bovine LPO. Thus, we could show that phenolic substances are suitable to regenerate the peroxidase activity in human saliva samples after H 2 O 2 -induced inactivation. Conclusion The studies provide new insights into the effect of pharmaceutical relevant polyphenols on salivary peroxidase activity and thus, suggest this enzyme as a new target for the prevention and therapy of oral inflammatory diseases.

Background The herbal medicinal product Myrrhinil-Intest®, a combination of myrrh, chamomile flower extract and coffee charcoal is used for the maintenance therapy of inflammatory bowel disease. In vitro studies revealed that the herbal components influence chemokine signaling of human macrophages as part of an anti-inflammatory mechanism. However, the occurrence of synergistic effects remains unexplored. Aim The present study aims to investigate the synergistic effect of dual combinations of the plant extracts on the pro-inflammatory chemokine (CXCL13) release from activated human macrophages. Methods The single effect of myrrh, chamomile flower and coffee charcoal extract on CXCL13 release from lipopolysaccharide stimulated human macrophages was investigated using ELISA and IC50 values were determined. Budesonide served as positive control. To characterize the combined effect, IC50 values were used to prepare combinations of two plant extracts in different proportions to each other (3:5; 1:1; 5:3). Interpretation of the data was based on isobologram analysis and calculation of a combination index (CI). Results LPS-induced CXCL13 release from human macrophages was inhibited after treatment with myrrh (IC50 = 19 μg/ml), chamomile flower (IC50 = 82 μg/ml) and coffee charcoal (IC50 = 106 μg/ml) whereby the extent of inhibition was comparable to budesonide. All combinations of two plant extracts resulted in synergistic effects with varying magnitude (CI from 0.82 to 0.42). The combination of myrrh and coffee charcoal (ratio of 3:5) exhibited the strongest synergistic effect (CI = 0.42). Increasing amounts of coffee charcoal resulted in increased synergistic activity. Conclusion Synergistic effects between all plant components contribute to the anti-inflammatory activity of all dual combinations of the plant components and support the composition of the herbal combination.

In animals and humans, dioxygen and glucose are the main substrates used for generation of universal energy equivalents in form of adenosine triphosphate. Uptake, transport, and utilization of dioxygen are highly regulated as these processes might be disturbed by the release of O2-binding proteins from red blood and muscle cells and the formation of highly cytotoxic metabolites such as methemoglobin, free hemoglobin, free myoglobin, and free ferric protoporphyrin IX, shortly known as free heme. In healthy individuals, glucose metabolism is also well controlled because of numerous chemical side reactions of glucose, which affect most of all the metabolism of insulin-independent cells under hyperglycemic conditions. Moreover, mitochondria can be a source for reactive species and damaging reactions. In this chapter, an overview is given about basic mechanisms on how energy metabolism can be disturbed, protective systems are presented, and links are provided to diseases associated with deficits in energy supply.

In human organism, inflammatory response is closely linked to synthesis of acute-phase proteins and activation of the complement, coagulation, and contact systems. These mechanisms act supplementary to immune cells and help to find an adequate response against external and internal impacts. Special functions of acute-phase proteins and proteins of the complement system are described during immune response. Coagulation is crucial to stop unwanted blood release from leaky and injured blood vessels. The contact cascade plays a pivotal role in response to the presence of bacterial, collagenous, and other surfaces. The activation of the contact system contributes to extravasation of fluid into tissues. In this chapter, mechanistic and regulatory details of supplementary systems are specified, and interrelations to inflammatory response and development of disease states are additionally highlighted.

Cell and tissue damage and functional constraints are also characteristic to the kidney, liver, and spleen diseases. Chronic kidney disease and liver cirrhosis can lead to fatal dysfunction of these organs. Underlying pathogenetic mechanisms given so far are known for these disease scenarios. In sepsis, the imbalance between tissue-damaging processes and host's protecting systems is further shifted toward the destruction side. Because of considerable decline or exhaustion of protective mechanisms, a potentiation of some tissue-damaging agents may occur. Fatal clinical events are septic shock and multiple organ dysfunction syndrome.

Reactive species contribute to destruction of biological material. In this chapter, an overview of main properties, formation, and reactions of reactive species is given. These reactants are classified into dioxygen-derived species, nitrogen-based species, transition metal ion–containing species, and (pseudo)halogen-based species. In particular, superoxide anion radicals, hydrogen peroxide, hydroxyl radicals, singlet dioxygen, nitric monoxide, peroxynitrite, nitrogen dioxide, hypochlorous acid, and hypothiocyanite are characterized in detail. The involvement of copper and iron ions and transition metal ion complexes in destructive reactions is also reviewed. Special aspects of participation of reactive species in cell and tissue destruction are outlined as well such as differentiation in low- and high-reactive species, redox recycling, water radiolysis, and the role of enzyme-driven reactions in formation of reactive species. Major enzymatic and nonenzymatic protective mechanisms are discussed controlling reactive species in biological systems.

Long-lasting chronic disease scenarios are associated with severe cell and tissue damage and numerous functional constraints. These diseases result from an imbalance between tissue-damaging processes and host's protecting systems. Energy and nutrient deficits, ischemia, limitation or exhaustion of host's repair systems, and immunocompromised states contribute to the weakness of defense mechanisms. In general, this mismatch is favored by an improper lifestyle, genetic predisposition, environmental factors, and advanced age. In this chapter, the role of destructive processes is analyzed in selected disorders such as cardiovascular diseases, diabetes mellitus, neurodegenerative diseases, autoimmune diseases, respiratory tract diseases, digestive tract diseases, and cancer. In description of disease pathogenesis and peculiarities, the main focus is directed on the underlying mechanisms of disease progression as well as cell and tissue damage. The role of immune defense is evaluated as well.

The immune system protects our organisms against foreign pathogens and is activated by traumata and numerous destructive external and internal impacts. Basic mechanisms of innate and acquired immunity are described in this chapter. Special attention is directed on pattern recognition receptors and their ligands, deactivation of microbes by immune cells, the interplay between neutrophils and macrophages, and damaging of host's tissues by immune cells. Regulation of inflammatory response is viewed in detail with the focus on resolution of inflammation. Presentation of antigens to lymphocytes and formation of specific antibodies is also considered. Disturbances and limitations of immune regulation and their consequences to pathological processes are further topics. These disease-related issues include persistence of chronic inflammation, immunosuppression, autoimmunity, hypersensitivity reactions, and disturbances in removal of waste material by lysosomes. Potential reasons of tissues destruction in immune-related diseases are discussed.

Inflammatory bowel disease or irritable bowel syndrome are chronic gastrointestinal disorders which are associated with a lifelong therapeutic need. The disease results in physical, psychological, and social problems with an impact on partnership, sexuality, education, and career. Thus, the number of patients and health care professionals relying on traditional and complementary medicines and especially phytotherapy for the treatment of these chronic conditions is increasing over recent years. One traditional herbal medicinal product consisting of chamomile flower, myrrh, and coffee charcoal has been widely used in clinical practice within this indication area. Long-term experience and an increasing understanding of the pharmacological mechanisms substantiate its application and clinical effectiveness. Mainly the spasmolytic and anti-inflammatory effects provide a rationale for its therapeutic application. In addition, synergistic effects between the herbal components contribute to the overall effect of this medication.

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.

15 pages, 9 figures, 2 tables, Cytochrome c (cyt c) is a small hemoprotein involved in electron shuttling in the mitochondrial respiratory chain and is now also recognized as an important mediator of apoptotic cell death. Its role in inducing programmed cell death is closely associated with the formation of a complex with the mitochondrion-specific phospholipid cardiolipin (CL), leading to a gain of peroxidase activity. However, the molecular mechanisms behind this gain and eventual cyt c autoinactivation via its release from mitochondrial membranes remain largely unknown. Here, we examined the kinetics of the H2O2-mediated peroxidase activity of cyt c both in the presence and absence of tetraoleoyl cardiolipin (TOCL)- and tetralinoleoyl cardiolipin (TLCL)-containing liposomes to evaluate the role of cyt c–CL complex formation in the induction and stimulation of cyt c peroxidase activity. Moreover, we examined peroxide-mediated cyt c heme degradation to gain insights into the mechanisms by which cyt c self-limits its peroxidase activity. Bottom-up proteomics revealed >50 oxidative modifications on cyt c upon peroxide reduction. Of note, one of these by-products was the Tyr-based “cofactor” trihydroxyphenylalanine quinone (TPQ) capable of inducing deamination of Lys ϵ-amino groups and formation of the carbonylated product aminoadipic semialdehyde. In view of these results, we propose that autoinduced carbonylation, and thus removal of a positive charge in Lys, abrogates binding of cyt c to negatively charged CL. The proposed mechanism may be responsible for release of cyt c from mitochondrial membranes and ensuing inactivation of its peroxidase activity, This work was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept for the SysMedOS project, Deutsche Forschungsgemeinschaft (DFG) Grant FE-1236/3-1 (to M. F.), European Regional Development Fund (ERDF; European Union and Free State Saxony) Grants 100146238 and 100121468 (to M. F.), and a Xunta de Galicia postdoctoral scholarship (to L. M.)

The clinical efficacy of curcumin has not yet been established for the treatment of cancer, despite a large body of evidence from numerous preclinical studies suggesting the therapeutic potential of curcumin, particularly in a synergistic combination with paclitaxel. The main obstacle in using curcumin for adjunctive cancer therapy is its low bioavailability via oral administration.We assessed the efficacy and safety of intravenous curcumin infusion in combination with paclitaxel in patients with metastatic and advanced breast cancer.A randomized, double-blind, placebo-controlled, parallel-group comparative clinical study was conducted.A total of 150 women with advanced and metastatic breast cancer were randomly assigned to receive either paclitaxel (80 mg/mThe intention-to-treat (ITT) analysis revealed that the ORR of curcumin was significantly higher than that of the placebo (51% vs. 33%, p0.01) at 4 weeks of follow-up. The difference between the groups was even greater when only patients who had completed the treatment (61% vs. 38%, odds ratio ==2.64, p0.01) were included. A superior effect of curcumin vs placebo was observed in both patients who had completed the treatment and all patients included in the ITT analysis, 3 months after termination of the treatment. No other significant differences were observed between the curcumin and the placebo groups, except for fatigue (3 vs. 10 patients, respectively; odds ratio ==3.7, p = 0.05). However, the patients' self-assessed overall physical performance was significantly higher with curcumin than the placebo during the treatment and at the end of the follow-up, suggesting better tolerance in the curcumin group.Overall, treatment with curcumin in combination with paclitaxel was superior to the paclitaxel-placebo combination with respect to ORR and physical performance after 12 weeks of treatment. Intravenously administered curcumin caused no major safety issues and no reduction in quality of life, and it may be beneficial in reducing fatigue.This is the first clinical study to explore the efficacy and safety of administering curcumin intravenously in combination with chemotherapy in the treatment of cancer patients.

The haem protein lactoperoxidase (LPO) is an important component of the anti-microbial immune defence in external secretions and is also applied as preservative in food, oral care and cosmetic products. Upon oxidation of SCN(-) and I(-) by the LPO-hydrogen peroxide system, oxidised species are formed with bacteriostatic and/or bactericidal activity. Here we describe the formation of the inter(pseudo)halogen cyanogen iodide (ICN) by LPO. This product is formed when both, thiocyanate and iodide, are present together in the reaction mixture. Using (13)C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry we could identify this inter(pseudo)halogen after applying iodide in slight excess over thiocyanate. The formation of ICN is based on the reaction of oxidised iodine species with thiocyanate. Further, we could demonstrate that ICN is also formed by the related haem enzyme myeloperoxidase and, in lower amounts, in the enzyme-free system. As I(-) is not competitive for SCN(-) under physiologically relevant conditions, the formation of ICN is not expected in secretions but may be relevant for LPO-containing products.

While the etiology of Alzheimer's disease (AD) is still unknown, an increased formation of amyloid-β (Aβ) peptide and oxidative processes are major pathological mechanism of the disease. The interaction of Aβ with free heme leads to the formation of peroxidase-active Aβ-heme complexes. However, enzyme-kinetic data and systematic mutational studies are still missing. These aspects were addressed in this study to evaluate the role of Aβ-heme complexes in AD. The enzyme-kinetic measurements showed peroxidase-specific pH- and H2O2-dependencies. In addition, the enzymatic activity of Aβ-heme complexes constantly increased at higher peptide excess. Moreover, the role of the Aβ sequence for the named enzymatic activity was tested, depicting human-specific R5, Y10, and H13 as essential amino acids. Also by studying Y10 as an endogenous peroxidase substrate for Aβ-heme complexes, ratio-specific effects were observed, showing an optimal dityrosine formation at an about 40-fold peptide excess. As dityrosine formation promotes Aβ fibrillation while free heme disturbs protein aggregation, we also investigated the effect of Aβ-heme complex-derived peroxidase activity on the formation of Aβ fibrils. The fluorescence measurements showed a different fibrillation behavior at strong peroxidase activity, leading also to altered fibril morphologies. The latter was detected by electron microscopy. As illustrated by selected in vivo measurements on a mouse model of AD, the disease is also characterized by Aβ-derived microvessel destructions and hemolytic processes. Thus, thrombo-hemorrhagic events are discussed as a source for free heme in brain tissue. In summary, we suggest the formation and enzymatic activity of Aβ-heme complexes as pathological key features of AD.

Layer-by-Layer (LbL) coated microcarriers provide a multifunctional drug delivery system for therapeutic substances into specific cells. Inflammatory cells as polymorphonuclear leukocytes (PMNs) represent a particularly promising target for LbL-microcarriers transporting anti-inflammatory substances such as α1 -antitrypsin (AT). They facilitate a local, low-dose and time-controlled application of the assembled therapeutic to effectively inhibit destructive enzymes provided by PMNs. But besides therapeutic effects, microcarriers themselves are not expected to affect cell viability. Thus, the present study emphasizes the investigation of LbL-microcarriers regarding their necrotic or apoptotic influence on inflammatory cells. The detection of mitochondrial membrane potential changes, reporting the induction of cellular apoptosis, was completed by the detection of apoptotic changes of the nucleus and lactate dehydrogenase release reporting potential necrotic influences. Investigations of microcarrier interactions with HL-60 cells and blood-isolated PMNs show very low influence on necrosis and, in case of AT-functionalized microcarriers, even a prolonged maintenance of mitochondrial membrane potential underlining the high potential of LbL-microcarriers.

Several hydrolyzable tannins, proanthocyanidins, tannin derivatives, and a tannin-rich plant extract of tormentil rhizome were tested for their potential to regenerate the (pseudo-)halogenating activity, i.e., the oxidation of SCN– to hypothiocyanite –OSCN, of lactoperoxidase (LPO) after hydrogen peroxide-mediated enzyme inactivation. Measurements were performed using 5-thio-2-nitrobenzoic acid in the presence of tannins and related substances in order to determine kinetic parameters and to trace the LPO-mediated –OSCN formation. The results were combined with docking studies and molecular orbital analysis. The –OSCN-regenerating effect of tannin derivatives relates well with their binding properties toward LPO as well as their occupied molecular orbitals. Especially simple compounds like ellagic acid or methyl gallate and the complex plant extract were found as potent enzyme-regenerating compounds. As the (pseudo-)halogenating activity of LPO contributes to the maintenance of oral bacterial homeostasis, ...

We investigated in vitro the ability of a standardised olive leaf dry extract (Ph. Eur.) (OLE) as well as of its single components to circumvent the hydrogen peroxide-induced inhibition of the hypothiocyanite-producing activity of lactoperoxidase (LPO). The rate of hypothiocyanite (⁻OSCN) formation by LPO was quantified by spectrophotometric detection of the oxidation of 5-thio-2-nitrobenzoic acid (TNB). By using excess hydrogen peroxide, we forced the accumulation of inactive enzymatic intermediates which are unable to promote the two-electronic oxidation of thiocyanate. Both OLE and certain extract components showed a strong LPO-reactivating effect. Thereby an o-hydroxyphenolic moiety emerged to be essential for a good reactivity with the inactive LPO redox states. This basic moiety is found in the main OLE components oleuropein, oleacein, hydroxytyrosol, caffeic acid as well as in different other constituents including the OLE flavone luteolin. As LPO is a key player in the humoral immune response, these results propose a new mode of action regarding the well-known bacteriostatic and anti-inflammatory properties of the leaf extract of Olea europaea L.

Phospholipase A(2) is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA(2) activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA(2) inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA(2) and phospholipids, as well as the application of MALDI-TOF mass spectrometry for screening PLA(2) inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.

A traditional herbal medicinal product, containing myrrh, chamomile flower, and coffee charcoal, has been used in Germany for the relief of gastrointestinal complaints for decades. Clinical studies suggest its use in the maintenance therapy of inflammatory bowel disease. However, the pharmacological mechanisms underlying the clinical effects are not yet fully understood.The present study aims to elucidate immunopharmacological activities of myrrh, chamomile flower, and coffee charcoal by studying the influence of each plant extract on gene expression and protein release of activated human macrophages.The plant extracts effect on gene and protein expression of activated human monocyte-derived macrophages was investigated by microarray gene expression analysis and assessment of the release of pro- and anti-inflammatory mediators (TNFα, chemokine CXCL13, and interleukin-10) using an ELISA test system.The extracts of myrrh, chamomile flower, and coffee charcoal influenced gene expression of activated human macrophages within the cytokine/chemokine signaling pathway. Particularly, chemokine gene expression was suppressed. Subsequently, the production of CXCL13 and, to a minor extent, cytokine TNFα was inhibited by all herbal extracts. Chamomile flower and coffee charcoal extracts enhanced interleukin-10 release from activated macrophages. The observed effects on protein release were comparable to the effect of budesonide, which decreased TNFα and CXCL13 and enhanced interleukin-10 release.The components of the herbal medicinal product influence the activity of activated human macrophages on both gene and protein level. The induced alterations within chemokine/cytokine signaling could contribute to a positive effect on the immunological homeostasis, which is disturbed in patients with chronic intestinal inflammation.

The chlorite-based drug solution WF10 has been successfully applied to dampen strong inflammatory disease states and to improve wound healing processes. Howev‐ er, the molecular mechanisms of this drug are not well understood. This study is di‐ rected to investigate how WF10 and its components affect the expression of surface markers and sulphated proteoglycans and glycosaminoglycans in proinflammatorystimulated monocytes and macrophages. Human blood-derived macrophages were cultivated from monocytes in the presence of 50 U/ml granulocyte-macrophage colony-stimulating factor and activated by a mix‐ ture of 100 ng/ml lipopolysaccharide (LPS) and 10 ng/ml interferon γ (IFNγ). These cells were identified and characterised by their specific cell-surface receptors CD14, CD16, CD80, CD86, CD163, and CD206 using flow cytometry approaches. The sulpha‐ tion level of proteoglycans and glycosaminoglycans was assessed by the BlyscanTM dye-binding assay. The expression of the surface marker CD44, a proteoglycan with sulphated glycosaminoglycan side chains, was followed by antibodies against CD44. The binding of fluorescence-labelled hyaluronan to CD44 was also investigated by flow cytometry. All analyses were performed after incubation of monocytes and mac‐ rophages with WF10 or with its main components chlorite and chlorate. The drug substance WF10 inhibited the activation of LPS/IFNγ-stimulated human monocyte-derived macrophages. Among them are the diminished expression of proinflammatory surface markers, the inhibition of the expression of the hyaluronan receptor CD44, and the binding of hyaluronan to CD44. Further, the overall amount of sulphated proteoglycans and glycosaminoglycans was down-regulated by WF10. These in vitro experiments indicate that WF10 is able to inhibit the proinflammatory activation of macrophages. The results suggested that chlorite is the active principle in WF10 as chlorite caused principally the same changes in targets as WF10. The WF10 component chlorate inhibited only the overall sulphation level of proteoglycans and glycosaminoglycans and the binding of hyaluronan to CD44. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To sum up, WF10 is a promising tool to inhibit proinflammatory states of immune cells. The inhibition of activation processes in monocytes and macrophages by WF10 coincides well with the results about clinical application of WF10.

In this study several flavonoids were tested for their potential to regenerate the (pseudo-)halogenating activity (hypothiocyanite formation) of the heme peroxidases lactoperoxidase (LPO) and myeloperoxidase (MPO) after hydrogen peroxide-mediated enzyme inactivation. Several flavonoid subclasses with varying hydroxylation patterns (especially of the flavonoid B-ring) were examined in order to identify structural properties of efficient enzyme regenerators. Kinetic parameters and second-order rate constants were determined. A 3',4'-dihydroxylated B-ring together with C-ring saturation and hydroxylation were found to be important structural elements, which strongly influence the flavonoid binding and oxidizability by the LPO/MPO redox intermediates Compounds I and II. In combination with docking studies these results allow an understanding of the differences between flavonoids that promote the hypothiocyanite production by LPO and MPO and those that inhibit this enzymatic reaction.

Glycosaminoglycans (GAGs) of the extracellular matrix (ECM) contribute to the regulation of physiological processes by binding various immune-competent proteins. Due to their large structural diversity, the analysis of the binding properties and their functional consequences is challenging. The cytokine interleukin-8 (IL-8) is involved in the recruitment of neutrophils to inflammatory sites. Here, we investigated the interaction of heparin hexasaccharides and recombinant human IL-8, consisting of 77 amino acids using fluorescence and NMR spectroscopy. A dissociation constant of 2.0±0.4 μM was determined for the heparin-IL-8 complex, which is slightly higher than what has been found for chondroitin-6-sulfate (K(D)=1.4±0.4 μM) [Pichert, A.; Samsonov, S. A.; Theisgen, S.; Thomas, L.; Baumann, L.; Schiller, J.; Beck-Sickinger, A. G.; Huster, D.; Pisabarro, M. T. Glycobiology2012, 22, 134-145], suggesting an important role of the sulfate group at position 6 of the second ring in the disaccharide unit of the GAGs in this interaction. In addition, the influence of long-chain hyaluronan, chondroitin sulfate, and heparin on IL-8-induced chemotaxis and oxidative activity of neutrophils was examined. Only the incubation of heparin with IL-8 affected the IL-8-mediated chemotaxis of neutrophils. However, all investigated GAGs enhanced the IL-8-induced formation of reactive oxygen species in neutrophils, which is an entirely new finding. This work provides a representative example of how protein functions can be regulated by different GAGs of the ECM.

Polymorphonuclear leucocytes (PMNs) accumulate at inflammatory sites and contribute to host defence, regulation of the inflammatory process, and also to tissue injury. Upon activation, these cells release the serine proteases elastase, cathepsin G, and proteinase 3 that are involved in multiple processes such as microbicidal activity, penetration of PMNs through endothelium and adjacent connective tissue to inflammatory sites, and processing of various cytokines. Here, we compared the three serine proteases for their release from PMNs and their ability to interact with resting PMNs and the highly sulphated glycosaminoglycan heparin. Unlike elastase, proteinase 3 and cathepsin G were released from resting PMNs as evidenced by flow cytometry, confocal fluorescence microscopy, and activity measurements. While proteinase 3 binds heavily to surface targets on vital PMNs, cathepsin G and elastase interact preferentially with sulphated glycosaminoglycans. These data revealed a differentiated picture about the individual functions of the PMN serine proteases during inflammatory response.

Recently, the complex of cytochrome c (cyt c), a small heme mitochondrial protein, with mitochondria specific lipid cardiolipin (CL) was linked to the control of apoptotic cell death. Under apoptotic conditions cyt c - CL complex exhibits peroxidase activity, leading to the formation of CL- hydroperoxides. CL-hydroperoxides promote cyt c release to the cytoplasm, where cyt c participates in apoptosis pathway. However, not much known about the modifications of cyt c as a part of cyt c - CL complex. In the present work, the systematic study on the peroxidase activity of CL-cyt c complex in CL-containing liposomes was performed. CL-hydroperoxides stimulate cyt c peroxidase activity in the absence of hydrogen peroxide. Furthermore, in the presence of oxygen, CL-hydroperoxides are generated as the products of the CL - cyt c peroxidase reaction. During peroxidase cycle cyt c amino acid residues are modified by reactive unsaturated aldehydes including 4-hydroxy- and 4-oxo-nonenal, derived from CL- peroxidation. This leads to the consecutive cyt c inactivation and heme degradation accompanied by the release of free iron. The current study on CL-driven cyt c degradation and iron release probably plays an important role in the mitochondrial pathway of apoptosis induction.

Drug delivery into immune cells has high potential for the treatment of all kinds of inflammation, allowing a target-oriented transport of active agents. The advantage of this local drug release is the prevention of negative effects of systemic applications and low-dose application. Thereby, the phagocytotic capability of mature phagocytes is essential. Microparticles can be loaded with immune regulatory substances to control and terminate inflammatory processes. In this study, silica microparticles were co-incubated with monocyte/macrophage-like cells in order to determine phagocytotic particle uptake. The phorbol ester-triggered differentiation was proven by the increased expression of surface markers as phosphatidylserine and CD14 and enhanced lysosomal activity. Particle/cell co-incubation results in cell surface attachment followed by phagocytosis. Phagolysosomal ingestion could be determined by co-localization using fluorescence staining techniques. In contrast, no particle interaction with undifferentiated cells could be found. Under phagolysosomal conditions, multilayer degradation within 22 h could be shown, indicating a valuable carrier basis design for the time-controlled delivery of active agents. Subsequently, it can be assumed that a higher differentiation degree allows phagocytosis of microparticles, providing drug delivery into immuno-active cells.

The heme-containing enzyme myeloperoxidase (MPO) accumulates at inflammatory sites and is able to catalyse one- and two-electron oxidation reactions. Here it is shown that (–)-epicatechin, which is known to have numerous beneficial health effects, in low micromolar concentration enhances the degradation of monochlorodimedon (MCD) or the chlorination of taurine in a concentration-dependent bell-shaped manner whereas at higher concentrations it sufficiently suppresses the release of hypochlorous acid. Presented reaction mechanisms demonstrate the efficiency of micromolar concentrations of the flavan-3-ol in overcoming the accumulation of compound II that does not participate in the chlorination cycle. In case of MCD the mechanism is more complicated since it also acts as peroxidase substrate with very different reactivity towards compound I (3 × 10 5 M −1 s −1 ) and compound II (8.8 M −1 s −1 ) at pH 7. By affecting the chlorinating activity of myeloperoxidase (–)-epicatechin may participate in regulation of immune responses at inflammatory sites.

The radiation-initiated transformations of ceramide as a constituent of micelles were studied. An amide was identified as a ceramide γ-radiolysis product. Determination of other radiolysis products and data on the interaction of the FeSO4-H2O2-ascorbate system with ceramide lead to the conclusion that the amide is formed as a result of OH-induced fragmentation in the polar moiety of the substrate.

Elevated levels of polymorphonuclear leucocyte (PMN)-derived elastase, which is suggested as marker for inflammations in the male genital tract, correlate well with spermatozoa deterioration. PMN elastase caused a time- and concentration-dependent (up to a elastase concentration of 0.5 microg/mL) externalization of phosphatidylserine and intercalation of propidium iodide on human spermatozoa. There are apparently a limited number of target sites for elastase on spermatozoa surface, because the further enhancement of elastase amount did not fasten alterations in spermatozoa parameters. Analysis of flow cytometry data revealed that most spermatozoa were in a necrotic state after an exposure with elastase for 22 h. Some apoptotic cells were only detected at shorter incubation periods. Seminal plasma prevented in a concentration-dependent manner the PMN elastase-mediated loss of vitality of spermatozoa. We detected by blotting techniques large amounts of alpha(1)-antitrypsin in seminal plasma. This antiproteinase is known to inactivate elastase at inflammatory sites. Increasing concentrations of alpha(1)-antitrypsin prevented gradually spermatozoa deterioration induced by elastase. Thus, alpha(1)-antitrypsin contributes to an efficient protease/antiproteinase balance in seminal plasma. A disturbed balance will promote the development of chronic inflammations which can also be the reason for male infertility problems.