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1.
Mol Immunol ; 93: 266-277, 2018 01.
Article En | MEDLINE | ID: mdl-28860090

Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein.


Candida albicans/enzymology , Complement C3/antagonists & inhibitors , Fungal Proteins/physiology , Amino Acid Sequence , Binding, Competitive , Calcium Signaling/drug effects , Candida albicans/drug effects , Candida albicans/immunology , Cell Line , Complement C3/immunology , Complement C3/metabolism , Complement C3/pharmacology , Complement C3a/antagonists & inhibitors , Complement C3a/pharmacology , Complement C3b/antagonists & inhibitors , Complement C3b/pharmacology , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/pharmacology , HEK293 Cells , Humans , Interleukin-8/metabolism , Neutrophils/drug effects , Neutrophils/physiology , Opsonin Proteins/immunology , Peptide Fragments/metabolism , Phagocytosis/drug effects , Protease Inhibitors/pharmacology , Proteolysis , Receptors, Complement/antagonists & inhibitors , Receptors, Complement/metabolism , Virulence/immunology
2.
Transfus Med Hemother ; 44(1): 46-51, 2017 Jan.
Article En | MEDLINE | ID: mdl-28275333

BACKGROUND: Living donor liver transplantation (LDLT) is an option to expand the donor organ pool for patients with life-threatening diseases who cannot be supplied with a cadaver organ. Next to the donor risks, complications after ABO-incompatible LDLT (ABOi LDLT) in the recipient are subject to controversial discussion. Improvement in ABOi graft survival rates have been achieved with plasma treatment procedures (PTP) and immunosuppression but antibody-mediated rejection (AMR) and graft loss still occur. METHODS: Since 2008, we have prepared 10 patients for ABOi LDLT. Seven of the 10 patients for transplantation had hepatocellular carcinoma (HCC). RESULTS: All patients underwent PTP before and after ABOi LDLT as well as immunosuppression according to the treatment schedule. We did not use anti-CD20 monoclonal antibodies in the transplant setting. We transplanted 6 of 10 preconditioned patients. After 3 years, 5 of the 6 transplanted patients were still alive. CONCLUSION: Even if B-cell depletion with anti-CD 20 treatment in the setting of ABOi LDLT is commonly accepted, our center successfully administered only quadruple drug immunosuppression combined with PTP. Especially patients with HCC had a high titer increment also pre-transplantation and were at high risk for arterial thrombosis and graft loss.

3.
Immunol Lett ; 168(1): 13-21, 2015 Nov.
Article En | MEDLINE | ID: mdl-26306739

The opportunistic pathogenic yeast Candida albicans employs several mechanisms to interfere with the human complement system. This includes the acquisition of host complement regulators, the release of molecules that scavenge complement proteins or block cellular receptors, and the secretion of proteases that inactivate complement components. Secreted aspartic protease 2 (Sap2) was previously shown to cleave C3b, C4b and C5. C. albicans also recruits the complement inhibitor factor H (FH), but yeast-bound FH can enhance the antifungal activity of human neutrophils via binding to complement receptor type 3 (CR3). In this study, we characterized FH binding to human monocyte-derived macrophages. Inhibition studies with antibodies and siRNA targeting CR3 (CD11b/CD18) and CR4 (CD11c/CD18), as well as analysis of colocalization of FH with these integrins indicated that both function as FH receptors on macrophages. Preincubation of C. albicans yeast cells with FH induced increased production of IL-1ß and IL-6 in macrophages. Furthermore, FH enhanced zymosan-induced production of these cytokines. C. albicans Sap2 cleaved FH, diminishing its complement regulatory activity, and Sap2-treatment resulted in less detectable CR3 and CR4 on macrophages. These data show that FH enhances the activation of human macrophages when bound on C. albicans. However, the fungus can inactivate both FH and its receptors on macrophages by secreting Sap2, which may represent an additional means for C. albicans to evade the host innate immune system.


Aspartic Acid Endopeptidases/immunology , Candida albicans/immunology , Complement Factor H/immunology , Fungal Proteins/immunology , Integrin alphaXbeta2/immunology , Macrophage-1 Antigen/immunology , Aspartic Acid Endopeptidases/metabolism , Blotting, Western , CD11b Antigen/genetics , CD11b Antigen/immunology , CD11b Antigen/metabolism , CD11c Antigen/genetics , CD11c Antigen/immunology , CD11c Antigen/metabolism , CD18 Antigens/genetics , CD18 Antigens/immunology , CD18 Antigens/metabolism , Candida albicans/enzymology , Candida albicans/physiology , Cell Line, Tumor , Cells, Cultured , Complement Factor H/metabolism , Cytokines/immunology , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Fungal Proteins/metabolism , Host-Pathogen Interactions/immunology , Humans , Integrin alphaXbeta2/genetics , Integrin alphaXbeta2/metabolism , Macrophage-1 Antigen/genetics , Macrophage-1 Antigen/metabolism , RNA Interference
4.
Atheroscler Suppl ; 18: 112-8, 2015 May.
Article En | MEDLINE | ID: mdl-25936314

BACKGROUND: The influence of ATR-1-autoantibodies on antibody mediated rejection (AMR) is still discussed controversially. Here we demonstrate some aspects as to diagnostics, treatment, clinical relevance and graft outcome. METHODS: A total of 27 transplant recipients (6 heart, 16 kidney, 3 lung and 2 multi-organ) suffering from AMR and a control group without transplant (8 pre-Tx, 1 pregnancy and 16 autoimmune and haematological diseases) were studied. In total, 290 IA eluates and the corresponding patient serum samples before and after immunoadsorption (IA) were analysed. RESULTS: ATR-1-and ETR-auto-antibodies (aAB) were found only in 4.5% of sera previous to IA treatment by using ELISA, but could be detected in 42% of IA eluates. AB with very high titres (>1:8 to 1:256) in the eluate were found more frequently in heart than in kidney recipients. These strong aAB are clinically relevant and cause dysfunction or loss of the grafts. A quick and reliable diagnosis of the aAB is essential for successful application of the therapeutic possibilities, like removal of the pathogenic autoantibodies or the blockage of their actions. CONCLUSION: The use of eluates for antibody detection was more sensitive and more reliable than patient serum. Yet, the test results are only meaningful when AB titres are measured, as this allows for a quick statement about the actual antibody elimination. The removal of pathogenic aAB via IA is better than medication-based treatment.


Autoantibodies/blood , Graft Rejection/immunology , Organ Transplantation , Receptor, Angiotensin, Type 1/immunology , Acute Disease , Adult , Aged , Biomarkers/blood , Blood Component Removal , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Female , Graft Rejection/blood , Graft Rejection/diagnosis , Graft Rejection/therapy , Graft Survival , Humans , Immunosorbent Techniques , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Organ Transplantation/adverse effects , Predictive Value of Tests , Risk Factors , Time Factors , Treatment Outcome , Young Adult
5.
Eur J Med Chem ; 94: 132-9, 2015 Apr 13.
Article En | MEDLINE | ID: mdl-25765759

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO activity in human primary leukocytes with IC50 = 0.8-2 µM, we synthesized 48 systematically modified derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 = 29 nM. Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental compound 2 (IC50 = 0.21 µM), and only modest suppressive effects on 12- and 15-LOs were evident. Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact cells that warrants further preclinical evaluation as anti-inflammatory agent.


Benzoquinones/chemistry , Lipoxygenase Inhibitors/chemistry , Lipoxygenase Inhibitors/pharmacology , Neutrophils/drug effects , Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Chemistry Techniques, Synthetic , Drug Evaluation, Preclinical/methods , Humans , Inhibitory Concentration 50 , Intramolecular Oxidoreductases/metabolism , Lipoxygenase Inhibitors/chemical synthesis , Neutrophils/enzymology , Prostaglandin-E Synthases , Structure-Activity Relationship
6.
mBio ; 6(2): e00143, 2015 Mar 17.
Article En | MEDLINE | ID: mdl-25784697

UNLABELLED: Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. IMPORTANCE: Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor.


Adaptive Immunity/drug effects , Candida albicans/physiology , Farnesol/metabolism , Immunologic Factors/metabolism , Quorum Sensing , Virulence Factors/metabolism , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Gene Expression Profiling , Humans , Monocytes/drug effects , Monocytes/immunology , Neutrophils/drug effects , Neutrophils/immunology
7.
Biochem Pharmacol ; 91(4): 490-500, 2014 Oct 15.
Article En | MEDLINE | ID: mdl-25107704

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10-100nM) increases the vesicular pH in these cells. Archazolid (10nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000nM. Of interest, secretion of interleukin-1ß was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.


Cytokines/metabolism , Endoplasmic Reticulum/metabolism , Macrolides/pharmacology , Monocytes/drug effects , Vacuolar Proton-Translocating ATPases/metabolism , Base Sequence , Cell Line , DNA Primers , Dose-Response Relationship, Drug , Humans , Microscopy, Fluorescence , Monocytes/enzymology , Monocytes/metabolism , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction
8.
Biochim Biophys Acta ; 1840(9): 2961-9, 2014 Sep.
Article En | MEDLINE | ID: mdl-24905297

BACKGROUND: Subcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists. METHODS: FLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy. RESULTS: 5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca(2+)-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB4. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP. CONCLUSION: The cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging. GENERAL SIGNIFICANCE: We present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.


5-Lipoxygenase-Activating Proteins/metabolism , Arachidonate 5-Lipoxygenase/metabolism , Cell Nucleus/enzymology , Indoles/pharmacology , Leukotrienes/biosynthesis , Lipoxygenase Inhibitors/pharmacology , 5-Lipoxygenase-Activating Proteins/genetics , Arachidonate 5-Lipoxygenase/genetics , Calcimycin/pharmacology , Calcium Ionophores/pharmacology , Cell Nucleus/genetics , HEK293 Cells , Humans , Leukotrienes/genetics
9.
J Med Chem ; 57(13): 5638-48, 2014 Jul 10.
Article En | MEDLINE | ID: mdl-24920381

The anticarcinogenic and anti-inflammatory properties of curcumin have been extensively investigated, identifying prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), key enzymes linking inflammation with cancer, as high affinity targets. A comparative structure-activity study revealed three modifications dissecting mPGES-1/5-LO inhibition, namely (i) truncation of the acidic, enolized dicarbonyl moiety and/or replacement by pyrazole, (ii) hydrogenation of the interaryl linker, and (iii) (dihydro)prenylation. The prenylated pyrazole analogue 11 selectively inhibited 5-LO, outperforming curcumin by a factor of up to 50, and impaired zymosan-induced mouse peritonitis along with reduced 5-LO product levels. Other pro-inflammatory targets of curcumin (i.e., mPGES-1, cyclooxygenases, 12/15-LOs, nuclear factor-κB, nuclear factor-erythroid 2-related factor-2, and signal transducer and activator of transcription 3) were hardly affected by 11. The strict structural requirements for mPGES-1 and 5-LO inhibition strongly suggest that specific interactions rather than redox or membrane effects underlie the inhibition of mPGES-1 and 5-LO by curcumin.


Curcumin/analogs & derivatives , Lipoxygenase Inhibitors/pharmacology , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacology , Arachidonate 5-Lipoxygenase/metabolism , Curcumin/chemical synthesis , Curcumin/pharmacology , Humans , Lipoxygenase Inhibitors/chemical synthesis , Male , Mice , Monocytes/drug effects , Monocytes/metabolism , Peritonitis/drug therapy , Structure-Activity Relationship
10.
Eur J Med Chem ; 81: 492-8, 2014 Jun 23.
Article En | MEDLINE | ID: mdl-24871899

5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the design, synthesis and biological evaluation of novel series of ethyl 5-hydroxyindole-3-carboxylate derivatives that efficiently inhibit human 5-LO. SAR analysis revealed that the potency of compounds is closely related to the positioning of the substituents at the phenylthiomethyl ring. The introduction of methyl or chlorine groups in ortho- and ortho/para-position of thiophenol represent the most favorable modifications. Among all tested compounds, ethyl 5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (19) is the most potent derivative which blocks 5-LO activity in cell-free assays with IC50 = 0.7 µM, and suppressed 5-LO product synthesis in polymorphonuclear leukocytes with IC50 = 0.23 µM.


Arachidonate 5-Lipoxygenase/metabolism , Drug Design , Indoles/pharmacology , Lipoxygenase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Indoles/chemistry , Lipoxygenase Inhibitors/chemical synthesis , Lipoxygenase Inhibitors/chemistry , Molecular Structure , Structure-Activity Relationship
11.
PLoS One ; 9(5): e96015, 2014.
Article En | MEDLINE | ID: mdl-24789333

Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.


Candida glabrata/genetics , Candida glabrata/immunology , Candidiasis/immunology , Candidiasis/microbiology , Macrophages/immunology , Phagosomes/immunology , Animals , Candidiasis/metabolism , Cell Differentiation/immunology , Cell Line , Humans , Hydrogen-Ion Concentration , Intracellular Space/immunology , Intracellular Space/metabolism , Intracellular Space/microbiology , Lysosomes/immunology , Lysosomes/microbiology , Macrophage Activation/immunology , Macrophages/cytology , Macrophages/metabolism , Macrophages/microbiology , Mice , Phagosomes/metabolism , Phagosomes/microbiology , Signal Transduction
12.
J Med Chem ; 57(9): 3715-23, 2014 May 08.
Article En | MEDLINE | ID: mdl-24697244

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 µM), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention.


Arachidonate 5-Lipoxygenase/metabolism , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Cytokines/biosynthesis , Enzyme Inhibitors/chemistry , HEK293 Cells , Humans , Indoles/chemistry , Inhibitory Concentration 50 , Molecular Structure , Monocytes/drug effects , Monocytes/metabolism
13.
Med Mycol ; 52(3): 223-39, 2014 Apr.
Article En | MEDLINE | ID: mdl-24625675

Candida albicans is a well-adapted human commensal but is also a facultative pathogen that can cause superficial and systemic infections. Its remarkable capacity to thrive within the human host relies on its ability to adapt and respond to the local environment of different niches. C. albicans is able to cope with oxidative stress in a coordinated fashion via upregulation of different protective mechanisms. Here, we unravel the role of a family of glutathione peroxidase (GPx), designated Gpx31, Gpx32, and Gpx33, in oxidative stress resistance. We show that GPx activity in C. albicans is induced upon exposure to peroxides and that this enzymatic activity is required for full resistance to oxidative stress. The GPx activity relies on the presence of GPX31, with no apparent contribution from GPX32 and GPX33 during in vitro short-term (3 h) exposure to peroxides. However, a triple gpx31-33Δ/Δ mutant exhibited a more pronounced sensitivity than a single gpx31Δ/Δ mutant on solid media in the presence of oxidants, suggesting that GPX32 and GPX33 may be involved in long-term adaptation to oxidative stress. Interestingly, reintegration of a single allele of GPX31 was sufficient to restore the wild-type phenotype in both the single and triple mutants. We found that mutants lacking GPX31-33 were more susceptible to killing by phagocytic cells, suggesting that GPxs are required for full resistance to innate immune effector cells. Despite the sensitivity to oxidative stress and phagocytes, these mutants were not affected in their virulence in the chicken embryo model of candidiasis.


Candida albicans/drug effects , Candida albicans/physiology , Drug Tolerance , Glutathione Peroxidase/metabolism , Oxidants/toxicity , Oxidative Stress , Peroxides/toxicity , Animals , Candida albicans/enzymology , Candida albicans/genetics , Cells, Cultured , Chick Embryo , Gene Deletion , Glutathione Peroxidase/genetics , Humans , Macrophages/immunology , Macrophages/microbiology , Microbial Viability/drug effects , Virulence
14.
Cardiovasc Res ; 101(3): 522-32, 2014 Mar 01.
Article En | MEDLINE | ID: mdl-24368834

AIMS: The small molecule indirubin-3'-monoxime (I3MO) has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and neointima formation in vivo. The influence of I3MO on VSMC migration and vascular inflammation, two additional key players during the onset of atherosclerosis and restenosis, should be investigated. METHODS AND RESULTS: We examined the influence of I3MO on VSMC migration, with focus on monocyte-derived leukotrienes (LTs) and platelet-derived growth factors (PDGFs) as elicitors. Exogenous LTB4 and cysteinyl leukotrienes as well as LT-enriched conditioned medium of activated primary human monocytes induced VSMC migration, which was inhibited by I3MO. I3MO also blunted migration of VSMC stimulated with the PDGF, the strongest motogen tested in this study. Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC. Notably, I3MO not only interfered with the migratory response in VSMC, but also suppressed the production of pro-migratory LT in monocytes. Conditioned media from monocytes that were activated in the presence of I3MO failed to induce VSMC migration. In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range. CONCLUSION: Our study reveals a novel dual inhibitory mode of I3MO on LT-mediated VSMC migration: (i) I3MO interferes with pro-migratory signalling in VSMC and (ii) I3MO suppresses LT biosynthesis in monocytes by direct inhibition of 5-LO. These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.


Cell Movement/drug effects , Cysteine/metabolism , Indoles/pharmacology , Leukotrienes/metabolism , Myocytes, Smooth Muscle/drug effects , Oximes/pharmacology , Signal Transduction/drug effects , Arachidonate 5-Lipoxygenase/metabolism , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Female , Humans , Male , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Neointima/drug therapy , Platelet-Derived Growth Factor/metabolism
15.
Eukaryot Cell ; 13(1): 170-83, 2014 Jan.
Article En | MEDLINE | ID: mdl-24363366

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.


Candida glabrata/pathogenicity , Fungal Proteins/metabolism , Genome, Fungal , Macrophages/microbiology , Oxidative Stress , Phagocytosis , Calcium/metabolism , Candida glabrata/genetics , Candida glabrata/metabolism , Cell Line , Cell Wall/genetics , Cell Wall/metabolism , Fungal Proteins/genetics , Gene Deletion , Humans , Iron/metabolism , Macrophages/immunology , Macrophages/metabolism , Neutrophils/immunology , Neutrophils/microbiology , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Virulence/genetics
16.
J Infect Dis ; 209(4): 616-26, 2014 Feb 15.
Article En | MEDLINE | ID: mdl-24163416

BACKGROUND: Natural killer (NK) cells are innate lymphocytes with potent cytotoxic activity. Whereas activity of NK cells has been demonstrated against the fungal pathogens Aspergillus fumigatus and Cryptococcus neoformans, little was known about their interaction with Candida albicans. METHODS: Primary human NK cells were isolated from buffy coats, primed with a cytokine cocktail and used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry, and enzyme-linked immunosorbent assay. RESULTS: Human NK cells actively recognized C. albicans, resulting in degranulation and secretion of granulocyte-macrophage colony-stimulating factor, interferon γ, and tumor necrosis factor α . Uniquely, activation of NK cells was triggered by actin-dependent phagocytosis. Antifungal activity of NK cells against C. albicans could be detected and mainly attributed to secreted perforin. However, NK cells were unable to inhibit filamentation of C. albicans. Human polymorphonuclear neutrophils (PMNs) counteracted the proinflammatory reaction of NK cells by preventing direct contact between NK cells and the fungal pathogen. Activation of PMNs was enhanced in the presence of NK cells, resulting in increased fungicidal activity. CONCLUSIONS: Our results show a unique pattern of NK cell interaction with C. albicans, which involves direct proinflammatory activation and modulation of PMN activity. For the first time, phagocytosis of a pathogen is shown to contribute to NK cell activation.


Candida albicans/immunology , Cytokines/immunology , Killer Cells, Natural/immunology , Neutrophils/immunology , Phagocytosis/immunology , Cell Communication/immunology , Cell Degranulation/immunology , Cells, Cultured , Cytokines/metabolism , Humans , Killer Cells, Natural/microbiology , Lymphocyte Activation , Microscopy, Fluorescence , Perforin/immunology , Perforin/metabolism
17.
J Med Chem ; 56(22): 9031-44, 2013 Nov 27.
Article En | MEDLINE | ID: mdl-24171493

Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) is currently pursued as potential pharmacological strategy for treatment of inflammation and cancer. Here we present a series of 26 novel 2-aminothiazole-featured pirinixic acid derivatives as dual 5-LO/mPGES-1 inhibitors with improved potency (exemplified by compound 16 (2-[(4-chloro-6-{[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]amino}pyrimidin-2-yl)sulfanyl]octanoic acid) with IC50 = 0.3 and 0.4 µM, respectively) and bioactivity in vivo. Computational analysis presumes binding sites of 16 at the tip of the 5-LO catalytic domain and within a subpocket of the mPGES-1 active site. Compound 16 (10 µM) hardly suppressed cyclooxygenase (COX)-1/2 activities, failed to inhibit 12/15-LOs, and is devoid of radical scavenger properties. Finally, compound 16 reduced vascular permeability and inflammatory cell infiltration in a zymosan-induced mouse peritonitis model accompanied by impaired levels of cysteinyl-leukotrienes and prostaglandin E2. Together, 2-aminothiazole-featured pirinixic acids represent potent dual 5-LO/mPGES-1 inhibitors with an attractive pharmacological profile as anti-inflammatory drugs.


Arachidonate 5-Lipoxygenase/metabolism , Intramolecular Oxidoreductases/antagonists & inhibitors , Microsomes/enzymology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Thiazoles/chemistry , Animals , Arachidonate 5-Lipoxygenase/chemistry , Binding Sites , Drug Design , Humans , Hydrophobic and Hydrophilic Interactions , Inhibitory Concentration 50 , Intramolecular Oxidoreductases/chemistry , Lipoxygenase Inhibitors/chemistry , Lipoxygenase Inhibitors/metabolism , Lipoxygenase Inhibitors/pharmacokinetics , Lipoxygenase Inhibitors/pharmacology , Male , Mice , Models, Molecular , Peritonitis/chemically induced , Peritonitis/drug therapy , Prostaglandin-E Synthases , Protein Conformation , Pyrimidines/metabolism , Pyrimidines/pharmacokinetics , Structure-Activity Relationship , Zymosan/pharmacology
18.
Biochem Pharmacol ; 86(4): 476-86, 2013 Aug 15.
Article En | MEDLINE | ID: mdl-23623753

Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) possesses anti-inflammatory and anti-carcinogenic properties in vivo, and these features have been related to interference with multiple targets including XIAPs, NFκB, STAT-3, Akt and mTOR. However, interference with these proteins requires relatively high concentrations of embelin (IC50>4 µM) and cannot fully explain its bioactivity observed in several functional studies. Here we reveal human 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase (mPGES)-1 as direct molecular targets of embelin. Thus, embelin potently suppressed the biosynthesis of eicosanoids by selective inhibition of 5-LO and mPGES-1 with IC50=0.06 and 0.2 µM, respectively. In intact human polymorphonuclear leukocytes and monocytes, embelin consistently blocked the biosynthesis of various 5-LO products regardless of the stimulus (fMLP or A23187) with IC50=0.8-2 µM. Neither the related human 12- and 15-LO nor the cyclooxygenases-1 and -2 or cytosolic phospholipase A2 were significantly affected by 10 µM embelin. Inhibition of 5-LO and mPGES-1 by embelin was (I) essentially reversible after wash-out, (II) not impaired at higher substrate concentrations, (III) unaffected by inclusion of Triton X-100, and (IV) did not correlate to its proposed antioxidant properties. Docking simulations suggest concrete binding poses in the active sites of both 5-LO and mPGES-1. Because 5-LO- and mPGES-1-derived eicosanoids play roles in inflammation and cancer, the interference of embelin with these enzymes may contribute to its biological effects and suggests embelin as novel chemotype for development of dual 5-LO/mPGES-1 inhibitors.


Anti-Inflammatory Agents/pharmacology , Anticarcinogenic Agents/pharmacology , Arachidonate 5-Lipoxygenase/metabolism , Benzoquinones/pharmacology , Intramolecular Oxidoreductases/antagonists & inhibitors , Lipoxygenase Inhibitors/pharmacology , Microsomes/enzymology , Antioxidants/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Eicosanoids/antagonists & inhibitors , Free Radical Scavengers/pharmacology , Humans , Leukocytes/drug effects , Leukocytes/enzymology , Molecular Docking Simulation , Prostaglandin-E Synthases , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors
19.
Atheroscler Suppl ; 14(1): 33-8, 2013 Jan.
Article En | MEDLINE | ID: mdl-23357138

Immunological problems like preformed donor specific antibodies (DSA) or high degree of immunization complicate the transplantation (TX) and can limit the therapeutic success. Essential pillars of antihumoral therapy are the extracorporeal procedures like therapeutic plasma exchange (TPE) and high plasma volume immunoadsorption (IA). Both extracorporeal treatments have the ability to remove pre-existing or newly formed antibodies quickly and effectively. In the last 5 years a total of 27 patients, 12 heart transplant (HTX) patients, 3 patients after combined lung and heart transplantation (Lu/HTX) and 12 lung transplant (LuTX) patients were treated for prevention (6 patients) or in case of rejection (20 patients) with TPE, cascade filtration (CF) or IA. 6 HTX recipients and 5 LuTX recipients were tested positive for HLA-ab either donor specific or de novo HLA-ab. Altogether 7 patients were tested positive for non-HLA-ab. 6 patients were treated prior to TX with 1-3 TPEs. After TX we treated 24 patients initially with TPE and performed on average 4 TPEs (1-11) for antibody elimination. 22 of 27 treated patients survived with stable graft function. The extracorporeal procedures we performed are qualified for rescue therapy of antibody mediated rejection (AMR).


Blood Component Removal , HLA Antigens/immunology , Heart Transplantation/immunology , Immunity, Humoral , Isoantibodies/blood , Lung Transplantation/immunology , Translational Research, Biomedical , Blood Component Removal/methods , Germany , Graft Rejection/immunology , Graft Rejection/prevention & control , Graft Survival , Heart Transplantation/adverse effects , Heart Transplantation/mortality , Heart-Lung Transplantation/immunology , Humans , Immunosuppressive Agents/therapeutic use , Lung Transplantation/adverse effects , Lung Transplantation/mortality , Plasma Exchange , Treatment Outcome
20.
J Lipid Res ; 54(4): 923-35, 2013 Apr.
Article En | MEDLINE | ID: mdl-23349208

Despite their beneficial anti-inflammatory properties, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may increase the infection risk at high doses, likely by generating an immune-depressed state. To assess the contribution of different immune cell populations to the immunomodulatory fatty acid effect, we comparatively investigated several aspects of inflammation in human T-helper (Th) cells and monocytes. Both fatty acids, but DHA to a lesser extent compared with EPA, selectively and dose-dependently reduced the percentage of cytokine-expressing Th cells in a peroxisome proliferator-activated receptor (PPAR)γ-dependent fashion, whereas the expression of the cell surface marker CD69 was unaltered on activated T cells. In monocytes, both EPA and DHA increased interleukin (IL)-10 without affecting tumor necrosis factor (TNF)-α and IL-6. Cellular incorporation of EPA and DHA occurred mainly at the expense of arachidonic acid. Concomitantly, thromboxane B (TXB)2 and leukotriene B (LTB)4 in supernatants decreased, while levels of TXB3 and LTB5 increased. This increase was independent of activation and in accordance with cyclooxygenase expression patterns in monocytes. Moreover, EPA and DHA gave rise to a variety of mono- and trihydroxy derivatives of highly anti-inflammatory potential, such as resolvins and their precursors. Our results suggest that EPA and DHA do not generally affect immune cell functions in an inhibitory manner but rather promote pro-resolving responses.


Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/pharmacology , Monocytes/drug effects , T-Lymphocytes, Helper-Inducer/drug effects , Arachidonic Acid/pharmacology , Benzamides/pharmacology , Cells, Cultured , Humans , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Monocytes/metabolism , PPAR gamma/metabolism , Pyridines/pharmacology , T-Lymphocytes, Helper-Inducer/metabolism , Tumor Necrosis Factor-alpha/metabolism
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