35th International Winter Workshop

Association between the activation of immune system and citrulline, a biomarker of gut function, during anticancer therapy

Bartoušková M, Zezulová M, Vitásková D, Študentová H, Kujovská-Krčmová L, Solichová D, Adam T, Melichar B

Palacký University Medical School and Teaching Hospital, Olomouc, Czech Republic, and Charles University Medical School and Teaching Hospital, Hradec Králové, Czech Republic

(bohuslav.melichar@fnol.cz)

Although the gastrointestinal toxicity is one of the principal side effects of chemotherapy, the assessment of still relies mostly on the symptoms reported by the patient. In a prior study in patients with rectal cancer we have demonstrated that citrulline represents a promising biomarker of gastrointestinal toxicity. In an earlier study, we have demonstrated a decrease of circulating citrulline levels accompanied by an increase in urinary neopterin in rectal cancer patients treated with chemoradiation. Both citrulline and neopterin concentrations correlated with the radiation dose. In the present study, citrulline and neopterin concentrations were studied in breast cancer and germ-cell tumor patients during the administration of systemic chemotherapy. In patients with breast cancer treated with neoadjuvant dose-dense chemotherapy, citrulline concentrations were significantly decreased one week after the administration of doxorubicin/cyclophosphamide cycles and then during the weekly paclitaxel-based therapy. While serum neopterin was mostly stable, urinary neopterin concentrations were increased throughout the course of treatment. In patients with metastatic breast cancer treated with the combination of trastuzumab, pertuzumab and docetaxel, citrulline concentrations were significantly decreased after first cycle of therapy, but returned subsequently to pretreatment levels. Serum neopterin concentrations were increased compared to baseline throughout the course of the therapy. A decrease of circulating citrulline concentrations was also observed in patients with germ-cell tumors treated with cisplatin-based chemotherapy. These data confirm that citrulline represents a promising biomarker of gastrointestinal toxicity of chemotherapy. Decrease of circulating citrulline was associated with increased neopterin concentrations.

Effects of nanoparticles on cultured immune cells

Becker K, Herlin N, Schennach H, Fuchs D, Gostner JM

Division of Biological Chemistry, and Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, and Central Institute of Blood Transfusion and Immunology, University Hospital, Innsbruck, Austria; and Au Service des Photons, Atomes et Molécules-Laboratoire Francis Perrin, Gif-sur Yvette, France

(kathrin.becker@i-med.ac.at)

The growth of nanotechnology in the scientific field and the application of nanoparticles for various purposes have dramatically risen. These ultrafine particles represent an intermediate position between bulk materials and molecular structures and offer new economic, scientific and therapeutic opportunities. Because of their small 1-100 nm size, the physicochemical properties of nanomaterials may differ from standard bulk materials and may pose a threat to human health. Only little is known about the effects of nanoparticles on the human immune system [1]. In this study, we investigated the effects of silver (Ag), aluminiumoxide (Al₂O₃), carbonnanotubes (CNT), zincoxide (ZnO) and gold-doped TiO₂ nanoparticles employing the in vitro model of human peripheral blood mononuclear cells (PBMC) [2], cytokine-induced neopterin formation and tryptophan breakdown were monitored (n = 4). Furthermore a reporter-cell line, THP-1-blue, can detect the activation of the redox-sensitive transcription factor nuclear factor-κB (NF-κB) [3]. Additionally, the ability of neutralization or production of ROS can be monitored by using a cell-based antioxidant capacity assay (n = 3).

Treatment with CNT resulted in differential outcome in unstimulated and stimulated cells. Neopterin levels were significantly increased after CNT exposure (18.75 - 150 μg/ml) in unstimulated PBMC, while in PHA-stimulated cells additional CNT resulted in a decrease of neopterin levels [37.5-150μg/ml]. The same observations were found for kyn/trp levels. In THP1-blue cells CNT were able to raise NF-kB activation significantly in LPS-stimulated cells. Treatment with Ag showed a significant and dose-dependent inhibition of tryptophan breakdown and IFN-γ production in stimulated cells (p <0.05), whereas, Al₂O₃ nanoparticle treatment did not significantly influence IFN-γ production and further T-cell responses. TiO₂ and gold doped TiO₂ [9.37-150μ/ml] were able to increase neopterin production in both stimulated and unstimulated PBMC. However kyn/trp levels were only influenced in stimulated cells, where inhibited IDO activity could be observed. Also ZnO treatment resulted in differential effects. Lower concentrations (2.34-18.75 μg/ml) act predominantly anti-inflammatory by inhibiting neopterin production in both stimulated and unstimulated PBMC. Higher concentrations already influenced cell viability significantly and were able to induce NF-κB activation significantly. Interestingly, nano-sized ZnO particles showed an induction of ROS-formation whereas bulk material showed no influence on ROS formation at all. Importantly, results were similar for all different tested ZnO sizes (50nm and 100nm and bulk material) and therefore not size dependent.

Our results showed that not all types of nanoparticles had similar effects on PBMC and THP1-blue cells in vitro. Every type showed particular results and have to be explored in more detail. Additionally, for diagnostic or therapeutic purposes, we have to keep in mind that nanoparticle exposure act differently during healthy or inflammatory conditions.

*Support by the Austrian Research Funds (Project 25150-B13) is gratefully acknowledged.

[1] Becker K, et al. Food Chem Toxicol 2014;65:63-9.

[2] Jenny M, et al. Inflamm Res 2011;60:127-35.

[3] Schroecksnadel S, et al. J Biomed Nanotechnol. 2011;7:209-10.

Increased ratios of neurotoxic tryptophan catabolites in bipolar disorder

Birner A, Platzer M, Bengesser SA, Lackner N, Queissner R, Fellendorf F, Reinighaus B, Mangge H, Fuchs D, Kapfhammer HP, Schwarz M, Reininghaus EZ

Department of Psychiatry, Research Unit on Lifestyle and Inflammation-associated Risk Biomarkers, Clinical Institute of Medical and Chemical Laboratory Diagnostics, and Institute of Pathophysiology and Immunology, Medical University of Graz, Graz, Austria; and Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

(eva.schmidt@medunigraz.at)

The neurobiological underpinnings of bipolar disorder (BD) are under permanent investigation. Emerging evidence suggests that immune-inflammatory activity and tryptophan catabolites (TRYCATs) changes are involved in the etiology as well as in the course of BD. In a former report we showed increased kynurenine/tryptophan ratios in 76 euthymic BD patients. In this study we want to evaluate the ratios between the neuroprotective kynurenine acid (KynA) and the neurotoxic TRYCATs 3-hydroxykunurenine (3-HK). We took peripheral TRYCAT blood levels of 144 euthymic BD patients and 109 healthy controls. For statistical analysis a MANCOVA controlled for age, sex, body mass index, cardiovascular disease and smoking was performed. Targeted parameters were the ratios of KYN to 3-HK (Kyn/HK), KynA to 3-HK (KynA/HK) and KYN_KYNA (Kyn/KynA). Our results showed significant bias to neurotoxic TRYCAT 3-HK. BD patients showed decreased KynA/HK (F=16,157; p<.001) and Kyn/HK (F=11.198, p<001) ratios. If separated for gender, the results only remained significant in male individuals with BD.

The results give further evidence for the involvement of TRYCATs in the pathophysiology of BD. The increase of the neurotoxic catabolite 3-HK compared to the neuroprotective KynA might be another puzzle stone to the etiology of cognitive deficits in BD.

Altered tyrosine and tryptophan metabolism during the schizophrenia illness course

De Picker L, Fuchs D, Coppens V, Sabbe B, Morrens M

Collaborative Antwerp Psychiatric Research Institute, Faculty of Medicine, University of Antwerp, Antwerp, Belgium; Biocenter, Division of Biological Chemistry, Medical University, Innsbruck, Austria

(livia.depicker@gmail.com)

Recent data suggest that inflammation, which has been implicated in the pathophysiology of schizophrenia, can influence the function of the enzymes indoleamine 2,3-dioxygenase 1 (IDO1), which converts tryptophan (TRP) to kynurenine (KYN), and guanosine-triphosphate-cyclohydrolase-1 (GTP-CH1), which through production of tetrahydrobiopterin triggers the conversion of phenylalanine (PHE) to tyrosine (TYR), as well as the production of downstream biogenic amines [1,2].

50 schizophrenia patients and 50 age- and sex-matched healthy controls are enrolled in a prospective longitudinal study. Blood samples are drawn from patients upon admission for an acute psychotic episode (<48h of antipsychotic treatment) and in remission. Healthy controls are sampled twice with a 2-3 month interval. Assays include tryptophan, kynurenine and their ratio as index of IDO1 activity; and neopterin, PHE, TYR, and nitrite as markers of GTP-CH1 activity. In addition, symptom severity in patients was assessed with PANSS interview at both sample times.

Preliminary results (n=25 per group) show that during a psychotic episode, patients’ levels of KYN were lower than those of controls (1.3 versus 1.5 μmol/l, p=0.02), and normalized upon remission. KYN/TRP ratio correlated negatively with PANSS positive symptom subscale (r=-0.34, p=0.02). Repeated measures analyses demonstrated a significant effect of group on PHE/TYR, TRP and nitrite levels, as well as a significant time*group interaction for nitrite.

Patients demonstrate altered tryptophan and tyrosine metabolism compared to controls in psychosis, which normalizes in remission. Further analyses of inflammatory parameters (C-reactive protein and cytokines) are planned to assess these alterations in relation to inflammatory state.

[1] Capuron L, et al. Biol Psychiatry 2011;70:175-182

[2] Okusaga O, et al. PloS one 2014;9:e85945.

Dopamine regulates iron homeostasis and innate immune responses of macrophages to salmonella infection

Dichtl S, Haschka D, Demetz E, Nairz M, Aβhoff M, Seifert M, Berger S, Weiss G

Department of Internal Medicine VI, Medical University of Innsbruck, Austria

(stefanie.dichtl@i-med.ac.at)

Siderophores are catechol based compounds which can bind iron. Iron is an essential growth factor for mammalian cells and microbes. Based on previous observations, showing increased bacterial growth in the presence of catechols, we asked whether this may be referred to hormone mediated alterations of iron homeostasis.

We studied the effects of the catecholamine dopamine on the regulation of iron in BMDM obtained from C57Bl/6wt mice and littermates knocked out for lipocalin-2, a mammalian siderophore binding peptide. The in vivo effects of dopamine were studied in wt mice infected with the Gram negative bacteria Salmonella typhimurium (S.tm.).

Administration of dopamine to macrophages resulted in a dose dependent increase of heme oxygenase-1 and ferroportin expression, the latter being the major cellular iron exporter, which subsequently resulted in reduced intramacrophage iron concentrations. This effect could also be reproduced upon infection of macrophages with S.tm.. These effects were independent from the presence/absence of the siderophore binding peptide lipocalin-2. The in vivo administration of dopamine to mice infected with S.tm. resulted in an increased bacterial burden in liver and spleen as compared mice receiving solvent. This is linked to an increased delivery of iron to bacteria in the presence of dopamine along with an impaired pro-inflammatory immune response of macrophages. Our data demonstrate that dopamine may deteriorate the course of infection by promoting bacterial growth which can be a major concern from the treatment of patients with bacterial sepsis receiving catecholamines.

N-methyl-D-aspartate and adenosine receptor mediated effect of organic and inorganic mercury on SH-SY5Y cell viability*

Engin AB, Engin ED, Tsatsakis AM, Golokhvast K

Gazi University, Faculty of Pharmacy, Department of Toxicology, Hipodrom, and Ankara University, Biotechnology Institute, Tandogan, Ankara, Turkey; Center of Toxicology Science & Research, Medical School, University of Crete, Heraklion, Crete, Greece; and Scientific Educational Center of Nanotechnology, Far Eastern Federal University, Engineering School, Vladivostok, Russia

(abengin@gmail.com)

Although the molecular mechanisms mediating methyl mercury- (MeHg) or mercury chloride- (HgCl₂) induced neurotoxicity are not completely understood, several lines of evidence indicate that oxidative stress represents a critical event related to the neurotoxic effects elicited by the mercury compounds. MeHg significantly decreases mitochondrial activity and can cause an elevation in the intracellular, cytosolic free Ca⁺² concentrations. Increase of the extracellular levels of glutamate is proposed to be the primary event that triggers the elevation in intracellular free Ca⁺² via over-activation of N-methyl-D-aspartate (NMDA) receptors in neurons. The aim of this study was to clarify whether the severity of MeHg and HgCl₂ cytotoxicity could be altered by regulating glutamate signal-transmission through the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in SH-SY5Y human dopaminergic neurons.

To determine the effects of organic or inorganic mercury compounds on cell death, SH-SY5Y cells were exposed to increasing concentrations of MeHg and HgCl₂ in the presence and absence of L-Glutamine, for 24 or 48 h and cell viability was examined by MTT assay, determining the nitric oxide (nitrite + nitrate, NOx) concentrations and related-oxidative stress. In order to examine the contribution of NMDA and AMPA receptors to the cell viability, increasing concentrations of kynurenic acid (KynA) and adenosine receptor antagonist, caffeine were added to the medium in the presence and absence of L-Glutamine and interferon-gamma (IFN-gamma). Following exposure to MeHg (1, 2 and 5 μM) and HgCl₂ (1, 2 and 5 μM) at both 24h and 48h, there were concentration dependent decreases in cell viability and inversely correlated oxidative stress, either in the absence or presence of L-Glutamine. Increase in the NOx generation-related oxidative stress and decrease in cell viability were marked with MeHg treatment in a dose-dependent manner, following 48 h incubation period in the presence of L-Glutamine. KynA, caffeine or IFN-gamma significantly improved cell viability by decreasing oxidative stress when compared with the MeHg exposed SH-SY5Y cells. However, concurrent exposure to increasing concentrations of MeHg or HgCl₂ and IFN-gamma to KynA- or caffeine-containing medium markedly reduced cell viability and enhanced oxidative stress in the presence of L-glutamine.

These data demonstrate that MeHg was more toxic than HgCl₂ and exhibited glutamate- and dose-dependent cytotoxicity in SH-SY5Y human neuroblastoma cells. KynA, caffeine and IFN-gamma provide a significant protection against MeHg toxicity through NMDA and AMPA receptors competition.

*Supported by The Scientific and Technological Research Council of Turkey, 214S112.

Transcriptional and translational control are important mechanisms for the regulation of adipogenesis

Fromm-Dornieden C, von der Heyde S, Salinas-Riester G, Brenig B, Beissbarth T, Baumgartner BG

Institute of Veterinary Medicine, and DNA Microarray Facility Göttingen, University of Göttingen, Statistical Bioinformatics, Department of Medical Statistics, University Medical Center, Göttingen, Germany; and Department of Internal Medicine, Metabolic Diseases and Medical Molecular Biology, Paracelsus Private Medical University Salzburg, Salzburg, Austria

(bernhard_baumgartner@gmx.de)

Much is known about transcriptional control during adipogenesis, including early events. Many adipogenic factors have been identified by RNA analysis that regulate cell fate and control transformation of preadipocytes into mature adipocytes. This process confers massive changes in the structure of the cell leading to the formation of fat vacuoles. The orchestrated structural changes require tight control of the involved factors. However, up to 60% of genes are controlled at the translational level rather than at the transcriptional level. Translational control plays a pivotal role in many dynamic processes and we presume that it is also important for the control of adipogenesis, especially in the first hours after hormonal induction.

3T3-L1 preadipocytes were cultured to confluence and adipogenesis was induced by standard protocols with hormones [1]. Cells were harvested at time points T0 (without hormone treatment) and T6 (6 hours after hormonal induction) and polysome bound mRNAs were separated from unbound by gradients centrifugation. Pools of polysomal and unbound mRNA fractions were analysed by microarray analysis. Changes in translational activity were calculated by comparison of mRNA expression levels at different time points in pools. Selected candidate genes were verified by qPCR and Western blotting.

By use of velocity sedimentation, we found that there is a general increase in gene activity within several hours of adipogenesis [2]. We identified 43 genes with increased abundancy in the polysomal fraction and 2 genes with reduced abundancy in the polysomal fraction after first six hours of adipogenesis in 3T3-L1 cells. Interestingly, we found Ghrelin and Ifit1, a silencer of translation, to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (Cdkn1c, eIF4b, Hsf1, Irf6, Myc, Plekhn1, Polr2a, Rpl18, Rpl27a, Rpl6, Rpl7a, Rps18, Rpsa, Sema3g, Tbc1d22a, Tsc22d3), form part of ribosomes (L6, L7a, L18, L27a, Sa, S18, S15a pseudogene), act on the regulation of translation (eIF4b) or transcription (Hsf1, IRf6, Myc, Tsc22d3). Others act as chaperones (Bag3, Hspa8/Hsc70, Hsp90ab1) or in other metabolic or signals transducing processes.

We conclude that a moderate reorganisation of the functionality of the ribosomal machinery is a very important step for growth and expression control at the beginning of adipogenesis.

[1] Fromm-Dornieden C, et al. BMC Mol Biol 2012;13:9.

[2] von der Heyde S, et al. BMC Genomics 2014;15:381

Influence of acute vs. chronic immune stimulation on tryptophan metabolism and mood

Fuchs D, Becker K, Strasser B, Gostner JM

Division of Biological Chemistry, and Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria

(dietmar.fuchs@i-med.ac.at)

The human immune system not only is deeply involved in the defense response against invading pathogens and malignant development, it also interacts with biochemical pathways relevant for neurotransmitter biosynthesis: During acute innate and adaptive immune responses Th1-type cytokine interferon-γ (IFN-γ) induces enzyme GTP-cyclohydrolase 1 (GCH-1), which initiates the production of 5,6,7,8-tetrahydrobiopterin (BH₄) in various cells, whereas neopterin is formed instead in human monocyte-derived cells. BH₄ is cofactor of enzymes phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH). They all are important for the biosynthesis of neurotransmitters dopamine, noradrenaline and adrenaline via dihydroxyphenylalanine (L-DOPA), as well as serotonin. In parallel to GCH-1, enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and inducible nitric oxide (NO^. ) synthase (iNOS) are triggered that are important for host defense acting merely in an antiproliferative way. IDO-1 initiates the breakdown of tryptophan via the kynurenine pathway, the enzyme being suppressed by NO^. . Thus, during treatment of patients with pro-inflammatory cytokines like interfereons these neurotransmitters can increase and may contribute to mood enhancement, cognition and well-being as well as a fall of blood pressure due to vasodilation when BH₄ promotes iNOS.

In contrast to the initial phase of immune response during, e.g., an acute infection, in the prolonged course of an immune response, states of chronic activation can develop [1]. This can be the case in autoimmune syndromes or during malignant tumor disease. Then the influence on biochemical pathways that depend on cofactor BH₄ is drastically different from the initial phase. Prolonged formation of reactive oxygen species like superoxide anion (O₂^-) by NADPH-oxidase (NOX) wipes out antioxidant systems and destroys antioxidants compounds including BH₄. Consequently, the function of BH₄-dependent enzymes is disturbed and the production of the above-mentioned L-DOPA-derived neurotransmitters becomes diminished. In a similar way, NO^. is reacting with O₂^- and produces highly toxic vasoconstrictant peroxynitrite (ONO₂^- ), causing hypertension rather than vasodilation. At the same time, NO^. no longer inhibitsIDO-1 and the formation of the kynurenine pathway metabolites is increased at the expense of tryptophan. Supported by the oxidative milieu, serotonin becomes diminished and the risk of low mood and depression development is increasing.

In summary, the influence of the immune system on the neurotransmitter biosynthetic pathways involving BH₄ is bidirectional. High neurotransmitter levels can derive from the enzymatic pathways which activity goes up when BH₄ is produced at high rates, as it is the case when pro-inflammatory cytokines activate GCH-1 during acute immune response, whereas in the chronic phases of immunity, oxidative stress has developed, which diminishes BH₄ and the enzymes that require this particular cofactor.

[1] Gostner JM, et al. Redox Report 2013;18:88-94.

Effects of exhaustive aerobic exercise on biomarkers of immune activation and tryptophan metabolism in trained athletes

Fuchs D, Geiger D, Schauer M, Gatterer H, Burtscher M, Strasser B

Division of Biological Chemistry and Division of Medical Biochemistry, Biocenter, Medical University Innsbruck, Innsbruck, Austria; University for Health Sciences, Medical Informatics and Technology, Institute for Nutritional Sciences and Physiology, Hall in Tyrol, Austria; University of Innsbruck, Department of Sport Science, Medical Section, Innsbruck, Austria

(barbara.strasser@i-med.ac.at)

Exhaustive exercise can cause a transient depression of immune function. Earlier studies show a significant influence of physical exercise to increase neopterin concentrations in serum and urine [1,2]. Data suggests significant effects of immune activation cascades on the biochemistry of monoamines and amino acids such as tryptophan. Tryptophan can be metabolized through different pathways, a major route being the kynurenine pathway which is often systemically up-regulated when the immune response is activated. The present study was undertaken to examine the effect of exhaustive aerobic exercise on biomarkers of immune activation and tryptophan metabolism in trained athletes [3].

After a standardized breakfast 2 h prior to exercise, 33 trained athletes (17 women, 16 men) performed an incremental cycle ergometer exercise test at 60 rpm until exhaustion. After a 20 min rest phase, the participants performed a 20 min maximal time-trial on a cycle ergometer (RBM Cyclus 2, Germany). During the test, cyclists were strongly encouraged to choose a maximal pedalling rate that could be maintained for the respective test duration.

Serum concentrations of amino acids tryptophan, kynurenine, phenylalanine, and tyrosine were determined by HPLC and immune system biomarker neopterin by ELISA at rest and immediately post exercise. Intense exercise was associated with a strong increase in neopterin concentrations (p <0.001), indicating increased immune activation following intense exercise and that activation of indoleamine 2,3-dioxygenase-1 (IDO1) is involved in the degradation of tryptophan during exercise.. Exhaustive exercise significantly diminished tryptophan concentrations (- 12%; p <0.001) and increased kynurenine levels by (+ 6%; p = 0.022). Also phenylalanine to tyrosine ratios were lower after exercise as compared with baseline (p <0.001). The kynurenine to tryptophan ratio correlated with neopterin (r = 0.560, p<0.01). Thus, increased tryptophan catabolism by indoleamine 2,3-dioxygenase appears likely. Peak oxygen uptake correlated with baseline tryptophan and kynurenine concentrations (r = 0.562 and r = 0.511, respectively, both p <0.01).

[1] Tilz GP, et al. Immunobiology 1993;188:194-202.

[2] Sprenger H, et al. Clin Immunol Immunopathol 1992;63:188-95.

[3] Strasser B, et al., PlosOne 2016;11:e0153617.

Liquid chromatography–tandem mass spectrometry method for determination of homocysteine and other biologically important thiols in plasma

Galba J, Michalicova A, Parrak V, Kovac A

Institute of Neuroimmunology, Slovak Academy of Sciences; AXON Neuroscience SE; and Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy of Commenius University, Bratislava, Slovak Republic; and Department of Pharmacology and Toxicology, The University of Veterinary Medicine and Pharmacy, Kosice, Slovak Republic

(jaroslav.galba@gmail.com)

We have developed and validated a sensitive, reproducible and robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in rat plasma. Samples were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and deproteinized with trichloroacetic acid (TCA). Subsequently the thiols were derivatized with N-phenylmaleimide (NPM) and the derivatization products were purified by solid phase extraction on mixed hydrophilic lipophilic balanced copolymer Oasis HLB. Separation of thiols was achieved by using hydrophilic interaction liquid chromatography (HILIC) chromatography using the BEH Amide (2.1mm x 100 mm, 1.7 μm) column. The compounds were detected by SRM in the ESI+ mode. We used stable-isotope labeled thiols (gluthathione-¹³C,¹⁵N, D3-cysteine, D4-homocysteine, D2- cysteinylglycine) as internal standards. The method was validated in compliance with the FDA guideline. Excellent linearity was observed for all analytes over their respective concentration ranges with correlation coefficients r² > 0.99. The intra- and inter-day precision and accuracy were within ±20%. The analytical recovery for spiked plasma samples was in range from 92% to 108%. The limits of quantitation (LOQ) were 0.25 μmol/L for cysteinylglycine and homocysteine, 0.50 μmol/L for glutathione and 5 μmol/L for cysteine. Using this method, we analyzed plasma samples from transgenic rat model for tauopathy and detected significant changes of important antioxidant glutathione.

Probiotic supplements affect tryptophan metabolism and reduce the incidence of upper respiratory tract infections in trained athletes: a randomized, double-blinded, placebo-controlled trial

Geiger D, Schauer M, Gatterer H, Burtscher M, Fuchs D, Strasser B

Medical University Innsbruck, Biocenter, Innsbruck, Austria; University for Health Sciences, Medical Informatics and Technology, Institute for Nutritional Sciences and Physiology, Hall in Tyrol, Austria; University of Innsbruck, Department of Sport Science, Medical Section, Innsbruck, Austria

(barbara.strasser@i-med.ac.at)

Prolonged intense exercise has been associated with a transient suppression of immune function. This has been linked to an increased risk for upper-respiratory tract infections (URTI). Data indicate significant effects of immune activation cascades on the biochemistry of monoamines, such as tryptophan (TRP, the serotonin precursor). TRP can be metabolized through different pathways, a major route being the kynurenine (KYN) pathway which is often systemically up-regulated when the immune response is activated. The present study was undertaken to examine the effects of a probiotic supplement during 3 month of winter training on biomarkers of immune activation and TRP metabolism after exhaustive aerobic exercise and on incidence of URTI in trained athletes.

Thirty-three highly trained individuals were randomized to probiotic (n = 17) or placebo (n = 16) groups, and under double blind procedures, received probiotic (PRO: OMNi-BiOTiC^® Power, Allergosan, Graz, Austria) or placebo (control) daily for 12 weeks. We determined serum concentrations of and TRP and KYN and of phenylalanine and tyrosine by HPLC, and neopterin by ELISA (BRAHMS, Hennigsdorf, Germany) at baseline and after 12 weeks at rest and immediately post exercise. Weekly training and illness logs were kept. Twenty-nine subjects completed the study (n = 14 PRO, n = 15 control). For eligibility testing all subjects performed an incremental cycle ergometer exercise test at 60 rpm until exhaustion. After a 20 minutes rest phase, athletes (17 women, 16 men; mean age 26.7 years; average body mass index 22 kg/m², average peak oxygen uptake (VO₂max) 51.4 ml/kg/min) performed a 20-minute maximal time-trial on a cycle ergometer. Exhausting exercise was associated with a strong increase in neopterin levels and of indoleamine 2,3-dioxygenase 1 (IDO1) activity as is indicated by KYN/TRP (both p <0.01). After 12 weeks, post exercise serum concentrations of TRP remained unchanged in PRO but decreased by 10% in the control group (significant treatment effect, p <0.05), indicating reduced TRP breakdown rates in PRO. The proportion of subjects on placebo who experienced 1 or more URTI symptoms was 2.5-fold higher than those on PRO (control 0.90, PRO 0.35; p <0.05). Results show that daily supplementation with probiotics appears beneficial in reducing the incidence of URTI in athletes during training periods in winter [1]. It remains unclear whether this effect is related or not to the influence of exercise on tryptophan catabolism mediated by IDO1.

[1] Fuchs D, et al., The International Sport & Exercise Nutrition Conference (ISENC), 15.-17.12.2015, Newcastle upon Tyne, UK

Effect of N-chlorotaurine on neopterin production and tryptophan breakdown, analytical aspects

Geisler S, Nagl M, Schennach H, Fuchs D, Gostner JM

Division of Biological Chemistry and Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria

(simon.geisler@i-med.ac.at)

N-Chlorotaurine (NCT) is a long-lived oxidant and in the field of medicine well known for its antimicrobial activity. In vivo, NCT is produced by activated human monocytes and granulocytes and besides its killing activity in host defense, NCT controls the inflammatory response. It also seems to be involved in the termination of inflammation. Small concentrations of NCT cause a down-regulation of pro-inflammatory chemokines, cytokines and enzymes, as shown in several publications [1,2]. When focusing Tryptophan (Trp) and Kynurenine (Kyn) concentrations in cell culture supernatants from NCT treated cells, a dose dependent decline of Kyn and Trp with a simultaneous decrease of cell viability was observed [2]. Based on these findings, further experiments were conducted to examine the effect of NCT on Trp- and Kyn-concentrations: Trp and Kyn were added to supplemented RPMI 1640 medium and processed by an advanced HPLC method as described earlier [3]. Results showed a huge effect of 0.16 mM NCT on the stability of Trp and Kyn. Concentrations above 0.32 mM NCT seemed to obliterate all Trp in the medium, whereupon Kyn depletion was observed only at concentrations above 1.25 mM NCT.

Sample preparation of NCT treated specimen without trichloroacetic acid (TCA) showed a significant decelerated Trp-degradation. The same was partly true for Kyn-concentrations. Examining the effects of NCT on inflammation one should be aware that factors like cell culture medium (containing reducing compounds, activated aromatic compounds etc.), pH, storage (freezing/defrosting) and a high ability to react with several biological materials could influence the stability of NCT and further affect its immunomodulatory properties. Besides, NCT chlorinates amino acids in the medium, forming the corresponding N-chloro derivatives by transhalogenation [1], which leads also to a degradation of the analyzed metabolites [3]. This effect seems to be amplified through addition of trichloro acetic acid. Considering these factors, alternative preparation steps of NCT-treated samples need to be examined.

[1] Gottardi W & Nagl M. J Antimicrob Chemother 2010;65:399-409.

[2] Wirleitner B, et al. 2004;93;143-9.

[3] Laich A, et al. Clin Chem 2002;48:579-81.

Neopterin measurements in human saliva

Geisler S, Schnell T, Gostner JM, Fuchs D

Division of Biological Chemistry, and Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, and Department of Psychology, Leopold Franzens University, Innsbruck, Austria

(dietmar.fuchs@i-med.ac.at)

The determination of neopterin concentrations allows a sensitive insight into the cellular immune status of patients that is relevant for judgment of disease activity and can predict prognosis in patients suffering from, e,g., HIV-1 infection or malignant tumor diseases. Neopterin concentrations can be measured in various body fluids such as serum, plasma, saliva, cerebrospinal fluid or urine. The information available on neopterin measurements in saliva is still limited and available data are often conflicting, e.g., neopterin measurements in saliva of patients with HIV-1 infection are less able to distinguish them from healthy controls as compared with in urine [1,2]. Moreover, saliva and serum neopterin concentrations were found not to correlate with each other [3]. From these studies it appeared that salivary neopterin concentrations are rather unrelated to systemic diseases processes. However, they were well able to reflect activities of local pathologic processes such as periodontitis when higher salivary neopterin was reported to occur in patients with periodontitis [4] and also in gingival crevicular fluid in patients with aggressive periodontitis [5].

In a study trying to link pteridine metabolism and mind [6] we measured saliva neopterin concentrations by ELISA (BRAHMS, Hennigsdorf, Berlin). In addition, also cortisol levels were monitored by ELISA (IBL, Hamburg). The experiment was based on the Trier Social Stress Test for groups and was performed in 50 individuals of whom saliva specimens were collected by Salivettes (Sarstedt) Saliva samples taken at baseline, after stressor 1 (public speaking task, framed as part of job interview), after stressor 2 (mental arithmetic task, count backwards in steps of 16 from 7582, as fast as possible) and four times during recovery period. When comparing neopterin concentrations throughout the study, it turned out that they were highly correlated although there was a time-dependent change of levels with a strong increase at the begin of the study and declining thereafter throughout all recovery episodes [6]. Correlation coefficients ranged from 0.502 (p = 0.002) to 0.856 (p <0001) in the stress-phase (time points 1-6) and were little less closely related when results were compared with the last recovery phase ranging from 0.326 (p = 0.055) to 0.846 (p <0001). Different results were obtained for cortisol levels when results of only 1 out of 15 time points correlated with each other significantly, and only time point 4 revealed significant correlations with the other time points ranging from 0.332 (p = 0.055) to 0.583 (p <0.001). Results imply that the measurements of saliva neopterin concentrations using Salivettes reveal quite stable results better than cortisol. Interestingly, measurements also imply that the strengths of correlations of cortisol levels gives opposite results as compared with neopterin levels.

[1] Vrecko C, et al. Clin Chem 1990;36:1379-80.

[2] Nishanian P, et al. Clin Diagn Lab Immunol 1998;5:507-12.

[3] Evans MR, et al. Clin Chem 1995;41:950-1.

[4] Vrecko K, et al. Clin Chim Acta 1997;268:31-40.

[5] Ozmeriç N, et al. J Periodontol 2002;73:720-5.

[6] Schnell T, et al. Pteridines 2015;26:130

Neopterin generation during inflammation and oxidative stress following high impact sport

Gieseg SP, Lindsay A, Draper N, Lewis J, Carr S, Cross, S, Peterson C

Sch. Biological Sciences, University of Canterbury, Christchurch, New Zealand; College of Life and Natural Sciences, Uni. Derby, United Kingdom; Canterbury Health Laboratories, Christchurch, New Zealand; School of Sport & Physical Education, University of Canterbury, New Zealand; and Dept Radiology, Uni Otago Christchurch, New Zealand

(steven.gieseg@canterbury.ac.nz)

The measurement of urinary neopterin and total neopterin appears to provide a good measure of the exercise and impact stress an individual athlete has been exposed to during the course of a competition [1]. Muscle damage is thought to promote inflammation and oxidative stress through activation of macrophages leading to the observed rise in urinary total neopterin and neopterin. In this study we have examined the reaction between 7,8-dihydroneopterin and possible oxidants released during muscle injury to identify how urinary neopterin may be generated during muscle injury.

SCX ion exchange HPLC [2] analysis showed that iron ions, and myoglobin but not cytochrome c can oxidise 7,8-dihydroneopterin to significant levels of neopterin and xanthopterin. Cytochrome c oxidised the majority of the 7,8-dihydroneopterin to xanthopterin with very little neopterin generation.

Analysis of urine from rugby players post match and Mixed Martial Arts (MMA) fighters post competition showed that there was a correlation between urinary myoglobin and urinary neopterin and xanthopterin levels. Ice bath treatment of the MMA fighters post competition lowered the urinary myoglobin, neopterin and xanthopterin levels further demonstrating a link between myoglobin and neopterin generation.

Collectively the data suggests the release of myoglobin from damaged muscle tissues facilitates the oxidation 7,8-dihydroneopterin to neopterin and xanthopterin. The measurement of urinary neopterin may provide a measure of oxidative stress post injury to an athlete and potentially a trauma patient.

[1] Lindsay A, et al. Scand J Med Sci Sports 2015: in press

[2] Lindsay A, et al. Pteridines 2014;25:53-62.

7,8-Dihydroneopterin oxidation to neopterin in macrophages and atherosclerotic plaques

Gieseg SP, Roake JA, ChenA, Katouah H, Cross SP, Othman MI, Prebble H

School of Biological Sciences, University of Canterbury and Dept Radiology, Uni Otago, Christchurch, New Zealand; Dept Vascular Endovascular & Transplant Surgery, Christchurch Hospital, New Zealand; Chem. Dept, Umm Al Qura University, Saudi Arabia & Uni Technical MARA, Malaysia

(steven.gieseg@canterbury.ac.nz)

Neopterin has been extensively used as a clinical marker of immune cell activation in a variety of conditions including cardiovascular disease (CVD). Though cited often as the oxidation product of 7,8-dihydroneopterin, the mechanism and location of neopterin generation during inflammation is undetermined. We have examined the oxidant production in macrophages and atherosclerotic plaques to determine the mechanism of 7,8-dihydropenopterin oxidation to neopterin in vivo.

7,8-Dihydroneopterin inhibits the cytotoxicity of oxLDL to human monocytes, macrophages and human derived U937 cells [1]. In these cells, the cell death is dependent on the generation of intracellular oxidants generated in response to the oxLDL. The oxidative stress causes the complete loss of intracellular GSH leading to the oxidative inhibition of key thiol containing enzymes such as glyceraldehyde 3-phosphate dehydrogenase and mitochondrial aconitase [2] 7,8-Dihydroneopterin appears to inhibit the oxidant dependent cell death by scavenging the intracellular oxidants as demonstrated by the reduction in staining of cells by the superoxide sensitive dye DHE. The addition of oxLDL to either macrophages or U937 cells in the presence of 7,8-dihydroneopterin causes a significant rise in intracellular neopterin levels. The addition of phorbol 12-myristate 13-acetate (PMA) or γ-interferon to cultured human plaque (from endarterectomy surgery) resulted in firstly the generation of 7,8-dihydroneopterin, then neopterin. The addition of the NADPH oxidase inhibitor apocynin reduced the level of neopterin generated in the plaque. When given to oxLDL treated U937 cells, apocynin also inhibited the loss of GSH and cell death. Collectively these observations suggest that neopterin may be generated in vivo through the intracellular scavenging of superoxide by 7,8-dihydroneopterin.

[1] Gieseg SP, et al. Antioxidants and Redox Signalling. 2010; 13(10):1525-34.

[2] Katouah H, et al. Int J Biochem Cell Biol 2015;67:34-42.

Dinitrochlorobenzene (DNCB) modulates immunocompetence

Gostner JM, Becker K, Schennach H, Pease J, Fuchs D

Division of Medical Biochemistry, and Division of Biological Chemistry, Biocenter, Medical University of Innsbruck; and Central Institute of Blood Transfusion and Immunology, University Hospital, Innsbruck, Austria; National Heart and Lung Institute, Imperial College London, South KensingtonCampus, London SW7 2AZ, UK

(johanna.gostner@i-med.ac.at)

2,4-Dinitrochlorobenzene (DNCB) is chemical used widely in industry. It belongs to the group of sensitizers, thus chemicals that induce a type IV hypersensitivity reaction. Indeed, DNCB induces a sensitizing reaction very efficiently and is thus frequently used for the induction of contact dermatitis in animal models. We were interested in the effect of DNCB on tryptophan catabolism in human immune cells.

The effect of DNCB addition on tryptophan breakdown was analysed in human peripheral mononuclear (PBMC) cells that were stimulated or not with mitogen phytohemagglutinin A. Addition of DNCB to the stimulated cells lead to a significant decrease of indoleamine 2,3-dioxygenase-1 (IDO-1) activity, as indicated by lower Kyn/Trp, at concentrations of 100 and 200 nM DNCB. Interestingly, a slight but significant effect was observable also in unstimulated PBMC at the higher concentration. Cell viability, as measured by resazurin reduction, was not affected with all treatment concentrations.

To investigate the effect of DNCB on IDO-1 in unstimulated cells in more detail, we analysed the expression of IDO-1 in the myelomonocytic THP-1 cells after 48h treatment with increasing concentrations of DNCB [100 – 1000 nM]. DNCB treatment was able to induce expression of IDO-1 up to 4-fold in these cells without further stimulation, changes were significant with concentrations of 400 nM and above. Cell viability was not affected up to 800 nM, while treatment with 1000 nM DNCB lead to a decrease of about 20% compared to control cells.

Current data suggest that IDO-1 is affected by this chemical sensitizer already at very low concentrations (nanomolar range) both on expression and activation level. We hypothesize that metabolic changes in tryptophan metabolism precede cytotoxicity. Further investigations are needed to investigate whether IDO-1 mediated tryptophan breakdown may serve as an early indicator of immunotoxicity.

Low grade inflammation-induced changes in neurotransmitter precursor amino acids and their possible role as biomarkers in cancer-related depression

Hüfner K, OberguggenbergerA, Kohl C, Geisler S, Gamper E, Meraner V, Egeter J, Hubalek M, Beer-Sandner B, Sperner-Unterweger B, Fuchs D

Department of Psychiatry and Psychotherapy; Division of Biological Chemistry, Biocenter; Department of Gynecology and Obstetrics and Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria

(katharina.huefner@i-med.ac.at)

Cancer patients often suffer from major depression or depressive syndromes. Although it is well known that depressive symptoms can appear at any time during the course of an oncological disease, certain periods for instance time after diagnosis carry a higher risk. Reported prevalence rates differ widely (up to 60%), reflecting also diagnostic difficulties. Oncologists recognize depression in their patients in only 15 to 50% of afflicted cases and the percentage of patients who receive adequate therapy is even lower [1]. Consequently, this leads to a reduced quality of life. Furthermore, impaired compliance/adherence and consequently a poorer prognosis of the oncological disease are discussed in this context.

A possible connection between chronic inflammation and the development of depressive symptoms has received increasing attention during the last 10 years. Elevated biomarkers of inflammation, including pro-inflammatory cytokines and neopterin, have been found in depressed patients, and administration of inflammatory stimuli has been associated with the development of depressive symptoms. Furthermore data have demonstrated that inflammatory cytokines can interact with pathways known to be involved in the development of depression, such as the monoamine metabolism. In the current study we investigated the association of neurotransmitter precursor amino acids with a diagnosis of depression or state anxiety in 154 subjects suffering from breast cancer (BCA(+)), depression (DPR(+)), both or neither. Neopterin, tryptophan and phenylalanine metabolites were analysed by HPLC or ELISA. The phenylalanine/tyrosine ratio (index of the catecholamine pathway) was associated with the factors “breast cancer” and “depression” and their interaction (all p < 0.001); it was highest in the DPR(+)BCA(+) group. The kynurenine/tryptophan ratio (index of the serotonin pathway) was significantly associated with the factors “breast cancer” and “state anxiety” and their interaction (p < 0.001, p = 0.026, p = 0.02, respectively); it was highest in the ANX(+)BCA(+) group. In BCA(+) patients kynurenine/tryptophan ratios correlated with severity of state anxiety (r = 0.226, p = 0.048, uncorrected) and phenylalanine/tyrosine ratios with severity of depressive symptoms (r = 0.376, p < 0.05, corrected) [2].

In conclusion, alterations of neurotransmitter precursor monoamines were observed in patients with breast cancer and they seem to influence mental health. Although the number of clinical trials using either psychotherapy or antidepressant medication for the therapy of depression in cancer patients is limited, good therapeutic options are available.

[1] Sperner-Unterweger B, et al. Wien Med Wochenschr 2015;165: 297-303.

[2] Hüfner K, et al. Psychoneuroendocrinology 2015;60:28-38.

A gatekeeper helix determines the substrate specificity of Sjögren-Larsson Syndrome enzyme fatty aldehyde dehydrogenase

Keller MA, Zander U, Fuchs JE, Kreutz C, Watschinger K, Mueller T, Golderer G, Liedl KR, Ralser M, Kräutler B, Werner ER, Marquez JA

Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria; Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK; European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France; Institute of General, Inorganic and Theoretical Chemistry and and Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria;MRC National Institute for Medical Research, the Ridgeway, Mill Hill, London, UK; andUnit of Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France

(markus.keller@i-med.ac.at)

In humans fatty aldehyde dehydrogenase (FALDH) is the major cellular detoxification system responsible for converting long chain fatty aldehyde – produced by tetrahydrobiopterin dependent cleavage of ether lipids by alkylglycerol monooxygenase – into their corresponding non-toxic fatty acids. ALDH3A2 is the gene coding for the NAD-requiring, membrane-bound fatty aldehyde dehydrogenase, that can be spliced in an endoplasmic reticulum and a peroxisomal variant. Mutations in this gene, that are affecting enzymatic functioning of FALDH, cause the Sjögren Larsson Syndrome; a severe, recessive-autosomal disorder with symptoms such as ichtyosis, mental retardation and spasticity. So far treatment options for this disease are marginal.

We succeeded in recombinant expression and subsequent affinity purification of active fatty aldehyde dehydrogenase, allowing to solve the first crystal structure of this enzyme. The fatty aldehyde dehydrogenase presents as a homodimer with two active sites with a large oligomerisation domain and the catalytic core being accessed by two binding funnels originating from diametral opposing positions of the protein, with one side binding NAD, and the other the substrate. At the entrance of the substrate funnel entrance we identified a novel structural element, a gatekeeper helix that restricts the funnels geometry and establishes a hydrophobic environment. Biochemical studies showed that this gatekeeper helix is responsible for the efficient conversion of long chain aldehydes, while there is no effect on the catalytic capacity for short chain substrates. Based on these data we were able to construct a model for fatty aldehyde dehydrogenase membrane interaction, co-factor handling, the catalytic mechanism as well as describing a mechanistic rationale for many Sjögren Larsson Syndrome causing missense mutations. While only about 20% of these are situated within the active site, the larger fraction of mutations impairs proper dimerization, protein folding and/or positioning of the gatekeeper helix.

Base on this structural study it is now possible to predict mechanistic details that are causative for the Sjögren Larsson Syndrome. In light of the large number of misfolding mutations, physicochemical stabilisation of the fatty aldehyde dehydrogenase enzymes would represent a promising strategy to at least partially recover residual enzymatic activity and to establish a novel treatment option to counteract some of the severe symptoms that Sjögren Larsson Syndrome patients are suffering from.

Immune activation, depression, and quality of life in patients with solid tumors

Kurz K, Holzner B, Klaunzner M, Willenbacher W, Fiegl M, Weiss G, Fuchs D

Department of Internal Medicine and Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria

(katharina.schroecksnadel@i-med.ac.at)

Patients with cancer often suffer from depression and experience an impaired quality of life. Immune-mediated disturbances of tryptophan and phenylalanine metabolism might be involved in the development of such „complications“ of tumor disease.

50 Patients with solid tumors and without infection or prediagnosed depression answered the EORTC QLQ C30 questionnaire and Beck depression inventory to estimate their quality of life and depression. Blood was drawn from patients and serum concentrations of tryptophan, kynurenine, phenylalanine and tyrosine were measured by HPLC, concentrations of neopterin by ELISA, and CRP, liver and renal parameters and blood counts were determined by automated tests. Trp/Kyn and Phe/Tyr ratio were calculated.

Immune-mediated tryptophan degradation coincided with phenylalanine accumulation. A high percentage of patients (38%) had depressive symptoms (14 patients with mild, 3 with moderate, 2 with severe depression). Patients with depression presented with significantly higher Phe/Tyr than patients without depression (p <0.001), furthermore also quality of life scores and emotional functionality were lower in patients with higher Phe/Tyr (both p <0.01). Patients with lower tryptophan concentrations reported about more fatigue (p <0.05). Immune-mediated disturbances of phenylalanine and tryptophan metabolism might be involved in the development of depression and might also contribute to deteriorated quality of life in patients with solid tumors.

Intestinal microbiome, neuroinflammation and neurodegeneration

Leblhuber F, Schuetz B, Fuchs D

Department of Neurological and Psychiatric Gerontology, Linz, Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria, Biovis Diagnostik MVZ GmbH, Limburg, Germany

(friedrich.leblhuber@liwest.at)

Neuroinflammation is a key component of Alzheimer’s disease (AD) and an early event in the pathogenesis of this most abundant form of dementia. Microglia represent key players of the inflammatory response in AD by pro-inflammatory molecules like tumor necrosis factor-α and interferon-γ. Neuroinflammatory processes are also involved in the neuronal degeneration in Parkinson’s disease (PD), the second most frequent neurodegenerative disease.

The brain-gut axis serves as communication system between the central nervous system and the gastrointestinal tract [1] with tryptophan as a key neurotransmitter precursor molecule. The gut microbial influence on tryptophan metabolism and the serotonergic system may be important in this regulation [2]. Age related changes in the intestinal microbiome with decreased diversity cause serotonin-related health problems in the elderly related to the ability of the gut microbiota to control tryptophan metabolism along the kynurenine pathway, thereby simultaneously reducing the fraction available for serotonin synthesis and increasing the production of neurotoxic metabolites due to silent inflammation. The enzymes of this pathway are immune and stress sensitive. Furthermore, neural processes in the gastrointestinal tract can be influenced by local serotonin decrease with subsequent signaling along the brain-gut axis influencing central nervous system neurotransmission. Treatment strategies with probiotics thus may have potential for serotonin-related disorders like anxiety, depression, as well as for cognitive decline and amyloidogenesis in AD.

PD affects all levels of the brain-gut axis including the central, autonomic, and enteric nervous system. Dysregulation of the brain-gut-axis in PD is associated with gastrointestinal manifestations frequently preceding motor symptoms, as well as with the pathogenesis of PD itself, supporting the hypothesis that the pathological process is spread from the gut to the brain. Excessive stimulation of the innate immune system resulting from gut dysbiosis and increased intestinal permeability induces systemic inflammation, initiating alpha-synuclein misfolding. Earlier diagnosis with a focus on peripheral biomarkers within the enteric nervous system may offer new therapeutic options modifying the gut microbiota, enhancing the intestinal barrier in PD. Interventions with probiotics in combination with traditional pharmacological treatments could influence the cascade of neuroinflammation and neurodegeneration in PD.

In conclusion, the role of the intestinal microbiome in neurodegeneration and the fundamental mechanisms underlying the relationship between diet, cognitive decline and motor dysfunction warrants further investigation.

[1] Bhattacharjee S, Lukiw WJ. Front Cell Neurosci 2013;7:153.

[2] Leblhuber F, et al. J Neural Transm (Vienna) 2015;122:1319-22.

Association between smoking and plasma tyrosine and kynurenine in schizophrenia

Mathai AJ, Fuchs D, Jyoti Kanwar J, Giegling I, Hartmann AM, Konte B, Friedl M, Rosenthal RN, Okusaga O, Rujescu D*, Postolache TP*

*Contributed equally and share senior authorship (ashwin.mathai@dc.gov)

Washington, DC, Baltimore, MD, Erie, PA, Tampa, FL, Denver, CO, Department of Psychiatry and of Epidemiology and Public Health, University of Maryland-Baltimore School of Medicine, Baltimore, MD; Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, Atlanta, GA; Division of Psychiatry and Behavioral Medicine, College of Human Medicine, Michigan State University and the Van Andel Research Institute, Grand Rapids, Michigan; Biocenter, Medical University, Innsbruck, Austria; University of South Florida, Tampa FL; Department of Psychiatry, Martin-Luther-University Halle-Wittenberg, Halle Germany; Veterans Integrated Service Network (VISN) 19, Mental Illness Research Education and Clinical Center (MIRECC), Denver, CO; Veterans Integrated Service Network (VISN), Mental Illness Research Education and Clinical Center (MIRECC), Baltimore, MD

(ashwin.mathai@dc.gov)

Cigarette smoking preferentially activates Th2 over Th1 immunity [1,2], thus increasing activity of phenylalanine hydroxylase (PAH) and decreasing indoleamine 2,3-dioxygenase (IDO) activity. Nicotine also induces tyrosine hydroxylase [3], the rate-limiting enzyme of catecholamine synthesis. We thus compared plasma levels of phenylalanine, tyrosine, kynurenine and tryptophan between smokers and non-smokers within a large sample of schizophrenia patients.

We measured phenylalanine, tyrosine, kynurenine and tryptophan levels using high performance liquid chromatography (HPLC) [4] in 920 subjects with SCID confirmed diagnosis of schizophrenia. ANCOVAs and logistic regression analyses were used to compare the neurochemical endpoints between current, past, and non- smokers.

Current smokers had lower tyrosine levels when compared to non-smokers (p = 0.02), and, even after adjusting for age, sex, BMI, chlorpromazine equivalent and Positive and Negative Syndrome Scale (PANSS) positive symptoms subscale, lower tyrosine levels predicted current smoking (p = 0.007). Smokers (past or current) had higher likelihood of decreased tyrosine levels (p = 0.03), and no difference in kynurenine levels when compared to nonsmokers. However, kynurenine levels were lower in current vs. past smokers (p = 0.04)

Decreased tyrosine levels in smokers may represent a treatment target in that subgroup of schizophrenia patients. Our finding our decreased kynurenine levels in current but no past smokers is consistent with smoking decreasing Th1 immunity and thereby suppressing IDO, reversible with quitting.

[1] Phaybouth V, et al. Am J Physiol Lung Cell Mol Physiol 2006;290:L222-31.

[2] Neurauter G, et al. Curr Drug Metab 2008;9:622-7.

[3] Hiremagalur B, et al. J Biol Chem 1993;268:23704-11.

[4] Neurauter G, et al. Clin Biochem 2013;46:1848-51.

Relationship between trait aggression and plasma phenylalanine tyrosine ratio in males with Toxoplasma gondii seropositivity

Mathai AJ, Cook T, Fuchs D, Giegling I, Hartmann AM, Konte B, Friedl M, Brenner LA, Lowry C, Groer MW, v

Washington, DC, Baltimore, MD, Erie, PA, Tampa, FL, Denver, CO, Department of Psychiatry and of Epidemiology and Public Health, University of Maryland-Baltimore School of Medicine, Baltimore, MD; Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, Atlanta, GA; Division of Psychiatry and Behavioral Medicine, College of Human Medicine, Michigan State University and the Van Andel Research Institute, Grand Rapids, Michigan; Biocenter, Medical University, Innsbruck, Austria; University of South Florida, Tampa FL; Department of Psychiatry, Martin-Luther-University Halle-Wittenberg, Halle Germany; Veterans Integrated Service Network (VISN) 19, Mental Illness Research Education and Clinical Center (MIRECC), Denver, CO; Veterans Integrated Service Network (VISN), Mental Illness Research Education and Clinical Center (MIRECC), Baltimore, MD

(ashwin.mathai@dc.gov)

We reported that trait aggression, an intermediate phenotype for suicidal behavior (Suicidal Self-Directed Violence, SSDV), was increased in Toxoplasma gondii-seropositive healthy individuals (who have an increased risk of SSDV) [1-3]. As immune activation, suppressing T. gondii in immunocompetent hosts, is known to decrease tyrosine (precursor of dopamine) [4], as T. gondii can synthesize dopamine, and because dopamine has been implicated in modulation of aggression, we sought to investigate the potential moderating effect of phenylalanine (Phe), tyrosine (Tyr) and their ratio (Phe:Tyr).

We measured plasma Phe and Tyr using High Performance Liquid Chromatography [5] in 1000 psychiatrically normal individuals (Munich region) as confirmed by SCID for DSM-IV-TR and determined T. gondii IgG titres using ELISA. Aggression was measured with FAF-(Fragebogen zur Erfassung von Aggressivitätsfaktoren). Statistical analysis was based on multiple linear regressions.

There was a significant correlation between Phe:Tyr ratio and Total-aggression (p = 0.03) and Self-aggression (p = 0.04) scores in T. gondii positive, but not negative males. Plasma Phe levels correlated positively with spontaneous-aggression (p = 0.01). We found a significantly higher spontaneous-aggression score in T. gondii positive males with Phe:Tyr in the upper quartile (p <0.03).

Higher aggression scores previously identified in T. gondii positive women, were now found in T. gondii positive men if they also had high Phe:Tyr ratio. It is possible that limiting dopamine synthesis may, in interaction with T. gondii seropositivity, contribute to trait aggression in males. This has potential implications for uncovering novel molecular targets for preventing violence in general, and SSDV in particular.

[1] Cook TB, et al. J Psychiatr Res 2015;60:87-94.

[2] Arling TA, et al. J Nerv Ment Dis 2009;197:905-8.

[3] Pedersen MG, et al. Arch Gen Psychiatry 2012;69:1123-30.

[4] Neurauter G, et al. Curr Drug Metab 2008;9:622-7.

[5] Neurauter G, et al. Clin Biochem 2013;46:1848-51.

Indoleamine 2,3-dioxygenase expression and immune response in breast cancer patients treated with primary chemotherapy

Melichar B, Čermáková P, Kujovská-Krčmová L, Bartoušková M, Solichová D, Ryška A

Palacký University Medical School and Teaching Hospital, Olomouc, Czech Republic, and Charles University Medical School and Teaching Hospital, Hradec Králové, Czech Republic

(bohuslav.melichar@fnol.cz)

Neoadjuvant (primary) chemotherapy is being increasingly used in patients with breast cancer. We assessed the association between the presence of tumor infiltrating lymphocytes (TIL), expression of indoleamine 2,3-dioxygenase (IDO), biomarkers of inflammatory response and pathological complete response (pCR) in a retrospective cohort of breast cancer patients treated with neoadjuvant chemotherapy. IDO expression and the presence of CD3+ TIL was evaluated immunohistochemically. IDO expression was classified as low in 72 cases, intermediate in 33 cases and high in 12 cases. The expression of IDO was significantly higher in human epidermal growth factor receptor (HER)-2 positive tumors. TIL counts were significantly increased in HER2-positive and triple negative (TN) tumors. No significant differences were observed in TIL between patients with high, intermediate or low IDO expression. Correlations were observed IDO expression and C-reactive protein (CRP) concentrations. No correlation was observed for intraturmoral TIL, while stromal TIL showed a weak, but significant correlation with urinary neopterin, CRP, neutrophil-to-lymphocyte ratio (NLR) and a negative correlation with absolute lymphocyte counts. Patients with pCR had significantly higher urinary neopterin concentrations, relative neutrophil counts, NLR and maximal and mean intraepithelial and stromal TIL counts and significantly lower relative and absolute lymphocyte counts. In a prospective cohort, circulating citrulline significantly decreased while urinary neopterin concentrations increased during the administration of neoadjuvant chemotherapy. Statistically significant correlations were observed between the investigated biomarkers. The decrease of hemoglobin concentrations was associated with complex and sometimes biphasic changes of iron metabolism. In conclusion, present data confirm that both high intraepithelial and stromal TIL counts are associated with pCR. Biomarkers of systemic inflammatory response were higher in patients who experienced pCR. Differences in IDO expression and TIL counts have been observed based on tumor phenotype, but IDO expression was not associated with the presence of TIL. Correlations were observed between stromal TIL counts and biomarkers of systemic inflammatory response. Citrulline represents a promising biomarker of gastrointestinal toxicity of neoadjuvant chemotherapy. In addition to decreased citrulline concentrations, the administration of intensive chemotherapy in breast cancer patients is accompanied by inflammatory response and complex changes of iron metabolism.

Pharmacometabolomics of tamoxifen therapy

Oberacher H, Arnhard K, Pitterl F, Sperner-Unterweger B, Oberguggenberger A, Hubelek M, Fiegl H, Marth C, Koal T

Institute of Legal Medicine and Core Facility Metabolomics, Medical University of Innsbruck, Innsbruck, Austria; Department of Psychiatry and Psychotherapy, Medical University of Innsbruck, Innsbruck, Austria; Department of Obstetrics and Gynecology, Medical University of Innsbruck, Innsbruck, Austria; Biocrates Life Sciences, Innsbruck, Austria

(herbert.oberacher@i-med.ac.at)

Metabolomics is an integral part of the broader field of systems biology and refers to the (un)targeted qualitative and (semi)quantitative analysis of the metabolome. The metabolome is the set of small molecular mass organic compounds (M_r <1500) found in a given biological medium. As metabolites are the ultimate downstream products of genomic, transcriptomic, and/or proteomic perturbations, changes in metabolite concentration and/or flux can reveal biologically relevant changes to the system. Such changes can be induced by xenobiotics. Thus, metabolomics represents a promising tool to study the molecular mechanisms of chemical-induced beneficial or toxicological effects.

Herein, we demonstrate the usability of pharmacometabolomics to get insides into tamoxifen therapy. Tamoxifen is a selective estrogen-receptor modulator which is used for the treatment of both early and advanced estrogen receptor positive breast cancer in pre- and post-menopausal women. Plasma samples obtained from 115 premenopausal women receiving 20 mg/d in an adjuvant setting were phenotyped with the AbsoluteIDQ® p180 Kit of Biocrates (Innsbruck, Austria). This enabled the quantitation of 125 metabolites, including amino acids, biogenic amines, acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, and sphingomyelines. In comparison to metabolite profiles of healthy controls (40 samples), distinct metabolic changes were observed, which included increased oxidative stress, changes in lipid composition, and alterations in markers related to immune modulation. In this way, we clearly demonstrated that pharmacometabolomics has the vast potential to expand our knowledge surrounding tamoxifen therapy.

Effects of complementary and alternative (CAM) interventions on immune activity in a patient with prior breast cancer: A time-series analysis approach

Ott M, Fritzsche K, Burbaum C, Hannemann J, Fuchs D, Schubert C

Psychoneuroimmunology Lab, Clinic for Medical Psychology, Medical University, Innsbruck, Austria

(michaela.ott@i-med.ac.at)

Cancer patients become increasingly interested in complementary and alternative medicine (CAM) apart from conventional medical treatment. A recent study revealed that 62% of female breast cancer survivors use complementary techniques, such as mind-body interventions, natural products, nutritional supplementation or energy-based techniques. Efficiency in reducing distress and associated inflammatory activity has been documented in some of these techniques, but there is still limited evidence. This study investigated the impact of CAM interventions (Tai Chi, Jin Shin Jyutsu, Energetic Healing, Physiotherapy) on the dynamic course of urinary neopterin levels in a prior breast cancer patient. For this purpose, a 49-year-old breast cancer patient diagnosed with a ductal mamma carcinoma 5 years ago collected her entire urine in 12-hour intervals for a period of 28 days (55 consecutive measurements), from 8 p.m. to 8 a.m. and from 8 a.m. to 8 p.m. Also in 12-hour intervals, she filled out questionnaires on emotional state, daily routine including used CAM techniques etc. Weekly, the patient was interviewed to gather evidence on the occurrence of past week’s personally meaningful everyday stressors. Mean concentrations of urinary neopterin over the whole observation period were 178 ± 22 μmol/mol creatinine. Cross-correlational analyses showed that all CAM interventions taken together in one variable significantly precede a decrease in urinary neopterin with a temporal delay of 72-84 hours (lag 6: r= -0.296; p ≤0.05). These results confirm the additive effect of various CAM interventions on immune function as proposed by other authors in this field. Since many patients with cancer have a chronically high level of inflammatory activity, we suggest that the decrease in urinary neopterin is associated with positive health consequences for the patient under study. This investigation showed that adequate research designs are needed to study effects of CAM interventions on immune function.

Oxyresveratrol modulates lipopolysaccharide- and mitogen-induced inflammatory responses in human PBMC and THP-1 cells

Palabiyik SS, Becker K, Halici Z, Bayir Y, Schennach H, Gostner JM, Fuchs D

Departments of Pharmaceutical Toxicology and of Biochemistry, Faculty of Pharmacy; Department of Pharmacology, Faculty of Medicine, Atatürk University, Erzurum, Turkey, Divisions of Biological Chemistry and Medical Biochemistry, Biocenter, Medical University, and Central Institute of Blood Transfusion and Immunology, University Clinics, Innsbruck, Austria

(sezinp@gmail.com)

Oxyresveratrol (OXY) is a polyphenolic phytoalexin produced by plants in Morus alba L and it is abundant in mulberry fruits and twigs (M. alba L.). The plants containing OXY have been reported to have several biological effects including anti-inflammatory and antioxidant effects [1]. During T helper (Th) type 1 immune response, interferon-γ (IFN-γ) which is primarily produced by activated T-cells, stimulates the enzyme indoleamine 2,3-dioxygenase (IDO) that converts the essential amino acid tryptophan to kynurenine. In parallel, IFN-γ stimulates the formation of the cellular activation marker neopterin. NF-κB is a key element in the regulation of immune activation and was measured by myelomonocytic THP-1-blue cells, a reporter-cell line, in this study [2]. Antioxidants are known to suppress Th1-type immune response via suppressing NF-κB activation and IFN-γ production [3]. The aim of the study was to investigate the effect on Th1-type immune response signaling of OXY in peripheral blood mononuclear cells (PBMC) and myelomonocytic THP-1 Blue cells. We investigated the effect of OXY on cell viability; tryptophan breakdown, neopterin production and activation of the redox-sensitive transcription factor nuclear factor-κB (NF-κB). Treatment with OXY has no cytotoxic effect in the tested concentration range (5 to 80 μM) after 48h of treatment on PMBC cells. 40 and 80 μM concentrations were able to suppress IDO activity significantly in mitogen stimulated PBMC and there is trend, but not significantly, of a decreasing effect in unstimulated control PBMC cells at 40 and 80 μM concentrations. Treatment with OXY also decreased neopterin production dose-dependently in mitogen stimulated PBMC cells at all concentrations (5 to 80 μM) and it is significant at doses >10 μM (p <0.05). Neopterin is also significantly decreased at 10 to 80 μM concentrations in unstimulated cells (p <0.05). In addition, treatment of the lipopolysaccaride (LPS)-stimulated NF-κB reporter cell line THP-1 Blue with OXY reduced NF-κB activity at lower concentrations (2.5 to 5 μM). These in vitro results suggests that OXY might have strong influences on immunological mechanism by interfering with IDO activity and neopterin production and on NF-κB expressions in isolated monocytic cells.

[1] Choi, et al. Chem Biol Interact 2015;239:153-63.

[2] Saha B, et al. Cytokine 2010;50:1-14.

[3] Schroecksnadel K, et al, Drug Metab Lett 2007;1:166-71.

Serum neopterin in the course during/after anti-tau protein vaccination in patients with Alzheimer’s disease – (Safety in clinical studies on tau immunotherapeutics)

Parrak V, Secnik P, Kontsekova E, Kovacech B, Kovac A, Katina S, Novak M

Axon-Neuroscience SE, Bratislava, and SK-Lab, Lucenec, Slovak Republic; and Masaryk University, Brno, Czech Republic

(parrak@axon-neuroscience.eu)

Alzheimer’s disease is still an incurable disorder with different course of cognition decline, deterioration of mental and physical status as well as very difficult period for the relatives. After discontinuation of more than 200 anti-amyloid clinical trials in last 2-3 decades the research is now focusing more on tau protein and development of anti-tau therapies [1].

We discovered that misfolded tau is a major component of neurofibrillary tangles (NFTs) in AD and other tauopathies. The research in Axon-Neuroscience led to the first phase of human clinical trials with anti-tau vaccine-AADvac1, which started in July 2013 in Austria. A total of 30 patients completed the study (12F/18M) with average age 69.1 ±8.1. In order to detect any side reactions after vaccine treatment we measured inflammatory markers neopterin and C-reactive protein (CRP) together with markers of neuronal injury-neuron specific enolase (NSE) and S100B protein. We detected significantly lower plasma levels of neopterin (6.09 ± 0.80 nM/l; n=27) in patients from AADvac-1 clinical study in compare to previously published data (16.2 ± 10.3 nM/l) from AD cohort [2]. Numerous studies reported a positive correlation between S100B levels in blood or cerebrospinal fluid and impaired neurological function following traumatic brain injury, intracerebral hemorrhage, stroke, extracranial injuries and others. In our study we didn’t find any significant changes in S100B and NSE levels. All measured values (7 month period with monthly interval) were within the reference ranges published previously [3,4]. We did not detect any other safety signals. Vital signs, ECG, blood and urine laboratory parameters, neurological and physical examination showed no significant differences and changes during the study. We can conclude that AADvac1 vaccine is safe, well tolerated and induces a robust immune response.

[1] Parrak V, et al. Pteridines 2004;15:50–154.

[2] Leblhuber F, et al. Clin Chem Lab Med 1999;37:429–431.

[3] Kleindiens A, et al. Cardiovasc Psych Neurol (Hindawi Publ Corp) 2010, ID 801295.

[4] Schaarschmidt H, et al. Stroke 1994;25:558-565.

Monitoring bladder cancer patients during intravesical BCG therapy: the prognostic value of urinary neopterin, sTNF-R75, sIL2R and serum neopterin and tryptophan breakdown

Pichler R, Fritz J, Culig Z, Fuchs D

Department of Urology, Department of Medical Statistics, Informatics and Health Economics, and Division of Biological Chemistry, Biocenter, Medical University Innsbruck, Austria

(renate.pichler@tirol-kliniken.at)

Intravesical instillation of bacillus Calmette-Guérin (BCG) for the treatment of high-risk, non-muscle invasive bladder cancer (NMIBC) represents one of the most successful tumor immunotherapies with 60-70% clinical response [1]. Nevertheless, 30-40% of patients confirm recurrence and 15% have progression to muscle invasive cancer with the necessity of cystectomy. As a delay in cystectomy affects the oncological outcome, an early identification of patients suited for bladder preservation with BCG therapy or radical cystectomy is essential in times of a worldwide BCG shortage [1-2].

Due to the fact that both biochemical pathways as well as the therapeutic efficiency of intravesical BCG therapy are induced and triggered by Th1-type cytokine IFN-γ [3-4], concentrations of serum neopterin and tryptophan degradation, urinary neopterin and sTNF-R75 and sIL2R could play a crucial role in monitoring patients during BCG therapy. A significant increase of serum and urinary neopterin concentrations 48 hours after each BCG instillation has already been shown in 71% of 30 patients [5]. 23 (9 women, 14 men) patients with a mean age of 68 years who were treated once a week with intravesically instilled BCG according to the empirical 6-weekly schedule were reviewed prospectively. Histology confirmed urothelial carcinoma in all patients. A second resection (2-6 weeks after initial surgery) was routinely performed in 7 patients (except CIS) to exclude persistent tumor and understaging by initial resection. Urine and blood samples were collected before therapy (baseline), during each instillation and 3, 6 and 9 months after BCG induction therapy. BCG failure was confirmed in 4 (17.4%) of 23 patients after a mean follow-up of 18 months. While serum (p = 0.012) and urinary (p = 0.003) neopterin, serum kynurenine (p = 0.015) and kynurenine-tryptophan ratio (Kyn/Trp, p = 0.005) concentrations were significant higher during BCG induction in BCG responders compared to BCG failure, levels of serum tryptophan (p = 0.154), urinary sTNF-R75 (p = 0.242) and sIL2R (p = 0.432) did not differ significantly based on oncologic outcome.

From this preliminary data obtained urinary and serum neopterin, kynurenine and Kyn/Trp seem to be valuable as novel monitoring parameter of BCG response, suggesting that a predominant Th1 cell-mediated immune response is necessary for effective BCG-induced antitumor effect. However, these preliminary results must be further studied in a larger prospective trial with sufficient statistical power prior to drawing any definitive conclusion.

[1] Babjuk M, et al. Eur Urol 2013;64:639-53.

[2] Biot C, et al. Sci Transl Med 2012, 6;4(137):137ra72

[3] Werner-Felmayer G, et al. Cancer Res 1990;50:2863-7.

[4] Maggi E, et al. J. Immunol 1992;148:2142-8.

[5] Mack D, et al. Eur J Cancer 1995;31A(6):1025-6.

Tryptophan breakdown and cognition in bipolar affective disorder

Platzer M, Lackner N, Fellendorf F, Birner A, Bengesser SA, Queissner, R, Kainzbauer N, Pilz R, Herzog-Eberhard S, Hamm C, Hörmanseder C, Maget A, Rauch P, Mangge H, Fuchs D, Zelzer S, Schwarz M, Reininghaus EZ

Department of Psychiatry, Research Unit on Lifestyle and Inflammation-associated Risk Biomarkers, Clinical Institute of Medical and Chemical Laboratory Diagnostics, and Institute of Pathophysiology and Immunology, Medical University of Graz, Graz, Austria; and Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; Ludwig-Maximilians-University Munich, Hospital Campus Grosshadern, Institute for Laboratory Medicine, Neurobiochemistry Research Group, Munich, Germany

(eva.reininghaus@medunigraz.at)

Bipolar disorder (BD) is often accompanied by various cognitive deficits pertaining to verbal memory, attention and executive functioning. Declining cognitive ability, e.g. memory disorders, attention deficits or impaired executive functions, substantially contribute to the high burden of disease in BD as the impact an individual’s overall psychosocial functioning.

In this investigation, 115 euthymic individuals with BD according to DSM-IV completed a cognitive test battery including the Trail Making Test-A/B (TMT-A/B), the d2 Test of Attention (d2 test), the Stroop test and the California Verbal Learning Test (CVLT). In addition, fasting blood samples were taken and serum levels of kynurenine and its metabolites hydroxykynurenine and kynurenic acid were analyzed. Subsequently ratios were formed from individual parameters. Patient data were compared with those of a mentally healthy control group (n=129).

In male participants with BD a higher hydroxykynurenine ratio was associated with overall better performance on the CVLT. Additionally, a significant negative correlation between hydroxykynurenine to kynurenic acid ratio and some CVLT-scores was found in male patients. Those associations were not present in mentally healthy male individuals. In the control group, however, a higher kynurenine to kynurenic acid ratio was associated with poor performance on the CVLT only in women. In addition, a positive correlation between kynurenic acid to hydroxykynurenine ratio and CVLT-scores was found. The findings at hand seem to suggest that in the metabolism of kynurenine a shift towards the hydroxykynurenine arm of the pathway– and therefore, ultimately, quinolinic acid – may be associated with poorer memory performance. Metabolites of the kynurenine pathway seem to be deserving of special scrutiny in the quest for identifying inflammatory markers that can predict the course of disease in BD. Gender specific differences are obvious and should be considered in research, diagnosis and therapy of BD.

Immunomodulatory effects of N-chlorotaurine on macrophages and dendritic cells during phagocytosis

Posch W, GostnerJM, Wilflingseder D, Nagl M

Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, and Center for Chemistry and Biomedicine, Division of Biochemistry, Medical University of Innsbruck, Innsbruck, Austria

(m.nagl@i-med.ac.at)

N-chlorotaurine (NCT), a long-lived oxidant with microbicidal properties produced by human granulocytes and monocytes, is known to downregulate pro-inflammatory cytokines in vitro and is thought to be involved in termination of inflammation [1,2]. Here we investigated its influence on cytokines of human macrophages and dendritic cells during phagocytosis. Monocyte-derived M1 Macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were challenged with Staphylococcus aureus ATCC 25923 at a ratio of 1:10 in RPMI plus 10% FCS medium. At the same time, NCT was 10-fold diluted in this mixture to final concentrations between 0.005% and 0.1%. Gene expression of pro- and anti-inflammatory cyto- and chemokines, i.e. IL-10, TGF-β, IL-1β, IL-6, IL-12, and TNF-α was relatively quantified by real-time RT-PCR using mRNA isolated from the cells after 1, 3, and 6 hours of incubation.

NCT at concentrations of 0.01% (0.55 mM), 0.03%, and 0.l% reduced the production of pro-inflammatory cytokines and chemokines after phagocytosis and an incubation time of 3h. Lower NCT concentrations had no influence. When bacteria were pre-treated with NCT for a sublethal time of 1 min before addition to the cells, these chlorine-covered bacteria induced a lower production of cytokines/chemokines than mock-treated ones. All these results were found in both cell types, MDM and MDDC. Our results confirm the anti-inflammatory properties of NCT, and its application in pharmacological concentrations (0.1%-2.0%) can be assumed to cause anti-inflammatory effects besides the microbicidal ones.

[1] Gottardi W, Nagl M. J Antimicrob Chemother 2010;65:399-409.

[2] Marcinkiewicz J, Kontny E. Amino Acids 2014;46:7-20.

Diagnostic and prognostic value of inflammatory parameters including neopterin in pneumonia, COPD and acute exacerbations of COPD

Pizzini A, Ziscka S, Sahanic A, Kurz K, Fuchs D, Weiss G, Bellmann-Weiler R

Department of Internal Medicine VI, and Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria

(alex.pizzini@i-med.ac.at)

Acute exacerbations and community acquired pneumonia (CAP) are severe complications in patients with chronic obstructive pulmonary disease (COPD), increasing hospitalization rates, morbidity and mortality (1). In this study inflammatory parameters including C-reactive protein (CRP), procalcitonin (PCT) and serum neopterin (NPT) were determined to evaluate their prognostic and diagnostic value, especially to differentiate between patients with CAP+COPD and with acute exacerbations of COPD (AECOPD) without pneumonia.

131 patients (CAP: n=65, mean age 65y, CAP+ COPD: n=29, mean age 71y, AECOPD: n=37, mean age 69y; overall 51 female and 80 male patients) were included in this study. CRP, PCT and blood counts were determined by routine automated tests, NPT concentrations by ELISA. The ratios of CRP to PCT and CRP to NPT levels were calculated and were used to identify cutoffs for discrimination between AECOPD and CAP (+/- COPD).

CRP and PCT levels were higher in patients with CAP (+/- COPD) compared to AECOPD patients (AECOPD vs. CAP p<0.001; AECOPD vs. CAP+COPD p <0.001). NPT levels were higher in patients with CAP compared to AECOPD patients (median 24.0 vs. 16.5 nM, p=0.027). CRP/NPT ratio was lower in AECOPD compared to CAP (+/- COPD) patients (AECOPD vs. CAP p<0.001; AECOPD vs CAP+COPD, p <0.001).

Positive correlations were found between duration of hospitalization and CRP, PCT and NPT concentrations. Patients who died within 30 days had higher NPT levels compared to survivors.

Calculation of the CRP/NPT-ratio suited well to discriminate between AECOPD and CAP, a CRP/NPT cutoff of 0.275 provided a sensitivity of 69% and a specificity of 83% to discriminate between the two diagnoses.

Our data confirm different inflammatory patterns in patients with AECOPD, CAP+COPD and CAP only. Our findings for the first time support the hypothesis that a fast discrimination between AECOPD and CAP+COPD is practicable by determining CRP and NPT levels. NPT levels may provide further prognostic information e.g. about duration of hospitalization as well as short term prognosis of patients.

Neuroimmunology of suicidal behavior: the upstream, the downstream and the modifiable

Postolache TT, Brenner L, Fuchs D, Brundin L

Department of Psychiatry and of Epidemiology and Public Health, University of Maryland-Baltimore School of Medicine, Baltimore, MD; Division of Psychiatry and Behavioral Medicine, College of Human Medicine, Michigan State University and the Van Andel Research Institute, Grand Rapids, Michigan; Biocenter, Medical University, Innsbruck, Austria; Veterans Integrated Service Network (VISN) 19, Mental Illness Research Education and Clinical Center (MIRECC), Denver, CO; Veterans Integrated Service Network (VISN) 5, Mental Illness Research Education and Clinical Center (MIRECC), Baltimore, MD

(teopostolache@gmail.com)

Clinical research suggests a link between inflammation and suicidal behavior. However, modifiable upstream triggers of inflammation and molecular pathways linking inflammation and suicidal behavior are not sufficiently understood. Latent neurotropic infections, such as with T. gondii have been associated with suicide attempts and, more recently, with intermittent phenotypes of impulsivity and aggression, intermediate phenotypes of suicidal behavior. Our very recent results also suggest that dopamine precursor levels in plasma interact with T. gondii seropositivity in predicting trait impulsivity and aggression. Our data suggest a potential role of certain tryptophan metabolites as mediators (quinolinic acid), protectors (picolinic acid), and effect modifiers (kynurenine) of T. gondii- attempt association, as well as the cytokine and kynurenine elevating effect of stress, a known suicide precipitant, will be discussed. Associations of suicidal behavior with very low levels of 25(OH) D will be discussed from a neuroimmune and neuroinfection standpoint. Reactivation of neurotropic infections as a result of activation of the kynurenine pathway will be proposed as a working hypothesis to be tested in future studies. Limitations of the concept of latent toxoplasmosis, and needed longitudinal and interventional designs will be addressed. Potential pharmacological targets for pilot studies and potential randomized controlled clinical trials will be discussed. In sum, our published and recent data suggest intersection of multilevel risk and protective factors and propose, to be tested in future experimental paradigms and clinical trials, much needed new intervention targets in suicide prevention based on neuroinfection and neuroimmunology.

Development of a kit to measure food antioxidant capacity using a novel chromometer

Priftis A, Tsatsakis AM, Konstantinopoulos C, Tzioumaks N,Kouretas D

Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece; Department of Forensic Sciences and Toxicology, Medical School, University of Crete, Heraklion, Greece; Coffee Island, Patras, Greece; and Polytech, Larissa, Greece

(dkouret@uth.gr)

Measuring the antioxidant capacity of foods is essential, as a means of quality control to ensure that the final product reaching the consumer, will be of high standards. Despite the already existing assays and kits with which the antioxidant activity is estimated, new, cheaper and faster ones are always sought. Therefore, we have developed a novel chromometer and combined it with a slightly modified DPPH assay, thus creating a kit that can assess the antioxidant capacity of liquids (e.g. different types of coffee, beer, wine, juices) in a quite fast and cheap manner. The accuracy of the chromometer was ensured by comparing it to a fully validated Hitachi U-1900 spectrophotometer, and a coefficient was calculated to eliminate the observed differences. In addition, a new, user friendly software was developed, in order to render the procedure as easy as possible, while allowing a central supervision of the obtained results.

Are serum neopterin levels helpful as prognostic marker in patients with decompressive craniotomy and bone flap donation?

Rainer V, Edlinger M, Ladner B, Schennach H, Mühlbacher A

Central Institute for Blood Transfusion and Immunology, General Hospital and University Clinics, and Department of Medical Statistics, Informatics and Health Economics, Medical University of Innsbruck, Innsbruck, Austria

(annelies.muehlbacher@tirol-kliniken.at)

After epidural hematoma or intracranial bleeding, decompressive craniotomy is performed to relief intracranial pressure rapidly. One option for cranial reconstruction is re-implantation of the autologous bone flap originating from craniotomy. For storage of bone tissue, screening for infectious diseases is mandatory. In the tissue bank of the University Hospital Innsbruck, situated in the blood bank, serum neopterin level assessment is part of these investigations. Neopterin is a biomarker for stimulated cellular immune response and is elevated after infection, during or following elective surgery, severe trauma, sepsis and cardiovascular disease.

The aim of this retrospective study of serum neopterin levels following craniotomy with storage of the bone flap, was to evaluate the difference in serum neopterin levels based on patient’s outcome-mortality or survival in the period of 3 months after surgery.

Between April 2010 and January 2015 in total 177 bone flaps from 152 different patients were stored at the tissue bank. 127 patients were included in the data analysis, 25 patients had to be excluded due to a missing neopterin measurement, missing information about the 3-months survival, or age under 12 years. Serum neopterin levels were measured using ELISA technique (Neopterin Screening EIA, BRAHMS GmbH, Hennigsdorf, Germany). The assay was performed and interpreted according to the manufacturer’s instructions. For most of the patients (114) the sample was drawn within 7 days after surgery, for the others the range was 8 to 19 days.

For the statistical analysis patients were grouped by serum neopterin level: group 1 normal level (<10.0 nmol/L); group 2 slightly elevated level (10.0- <20.0 nmol/L); group 3 strongly elevated level (≥20.0 nmol/L). “Normal” (<10.0 nmol/L) was defined according to blood donor screening. Blood donations with serum neopterin levels <10.0 nmol/L are allowed to be released for transfusion. Statistical analysis was done using SPSS version 21.

The 127 included patients had a mean age of 47.8 (standard deviation SD 17.5 years, range 12 to 83), 50 of them (39%) were female. Of all patients 39 (31%) died within 3 months. The reason for decompressive craniotomy was a traumatic brain injury in 56 (44%) and a malignant brain infarction in the rest (56%) of the cases. Concerning serum neopterin levels, 56 patients (44%) were in group 1, 48 (38%) in group 2, and 23 (18%) in group 3. Just about half of the patients had normal serum neopterin levels. The percentage of 3-month survival was 77% in group 1, 65% in group 2, and 61% in group 3 (p = 0.07, Mann-Whitney U test). There was a higher risk of mortality within 3 months after surgery (odds ratio of 1.9) for patients with elevated serum neopterin levels.

Of the 56 patients with traumatic brain injury 19 (34%) had normal serum neopterin levels, whereas 28 (50%) had slightly elevated and 9 (16%) strongly elevated levels. In the group of patients with malignant brain infarction, 37 (52%) presented with normal neopterin levels and 34 (48%) with elevated levels (20 in group 2 and 14 in group 3). In the trauma group the neopterin levels were significantly higher (Chi-squared test, p=0.049) and had, compared to the brain infarction group, a higher mortality risk (odds ratio of 2.1).

Despite a higher mortality risk for patients with elevated serum neopterin levels after decompressive craniotomy, serum neopterin levels do not seem to be really helpful as a prognostic marker for the attending physician.

Interactions of neutral pterin with the hydroxyl radical-studies using density functional theory

Reibnegger G

Institute of Physiological Chemistry, Center of Physiological Medicine, Medical University Graz, Graz, Austria

(gilbert.reibnegger@medunigraz.at)

Pteridines interact with free radicals, and may trigger free radical formation [1]. The occurrence of pteridine radicals has been hypothesized as intermediates in photobiological processes [2], and first results on the electronic nature of such systems are available [3].

Using reliable quantum chemical computations, first studies on putative reactions of the aggressive and highly reactive hydroxyl radical with neutral pterin were performed. Here, either abstraction of hydrogen atoms from pterin resulting in the formation of water and pterin radicals or direct attack of the free radical onto a pterin ring carbon atom, leading to hydroxylation, were studied.

All calculations were done in gas phase. Density functional theory at the UBecke3LYP/6-31G(d) level was used to search transition states for the studied reactions, as well as for initial geometry optimizations and frequency analyses. Final structures of the involved molecules and transition states were computed at the quite accurate UBecke3LYP/6-311+G(2d,p) level. Electron density, spin density and electrostatic potential (ESP) functions were obtained in cubes surrounding the molecules and employing a 192x192x192 grid. These calculations were done using G09W software (Gaussian Inc., Pittsburgh, PA, USA). Analysis according to the quantum theory of atoms in molecules (QT-AIM) is performed by the AIMAll package (TK Gristmill Software, http://aim.tkgristmill.com/). This software is also employed for building molecular graphs and graphical representation of the charge density gradient fields and the atomic basins of attraction. Electron localization functions (ELF) were computed using the free program DGRID (M Kohout, http://www2.cpfs.mpg.de/∼kohout/dgrid.html). Visualization of electron as well as spin density function, ESP, ELF and the Laplacian of the electron density function was done with AVS Express 5.3 software (Advanced Visual Systems Inc., Waltham, MA, USA).

The results demonstrate that an attack of hydroxyl radicals with abstraction of one of the three available hydrogen atoms bonded to nitrogen atoms is well possible, with intermediate formation of a well-defined transition state (TS; a stationary but not stable state on the energy hypersurface, verified by the occurrence of one negative vibrational frequency). Intrinsic reaction coordinate (IRC) computations starting from these TS demonstrate rather small energy activation barriers of about +2 kcal/Mol. The total reactions (hydroxyl radical and pterin yielding water and a pterin radical) are energetically strongly favourable with energy differences between the products and reactants of roughly -15 to -20 kcal/mol, depending on which pterin radical is formed. Spin density during the reaction is completely transferred from the hydroxyl radical onto the pterin ring system. Clearly, the driving force of these reactions is the transformation of neutral pterin and the highly reactive hydroxyl radical with its single electron (being localized exclusively to the oxygen atom), into stable water and a comparably stable and only moderately reactive pterin radical (with the single electron being delocalized over the whole molecular system).

In contrast, such hydrogen abstraction reactions cannot be confirmed for the hydrogens bonded to C6 and C7; rather, in these cases energetically favourable reactions are the respective hydroxylation reactions taking place at either C6 or C7. Energy barriers here appear negligible and total reaction energies are in the range of about -20 kcal/mol. Also in these cases, spin density is transferred to the pterin system, and the strongly delocalization of the single electron in each of the resulting 6- or 7-hydroxy-pterin radicals drives the reaction.

Future work will focus on other pterin systems (e.g., dihydro- and tetrahydro-structures) and other biologically interesting free radicals, as well as on the influences of solvents, pH, and biologically meaningful substituents at the pterin ring system.

[1] Öttl K, et al. Helv Chim Acta 2000;83:954-965.

[2] Lorente C, et al. Pteridines 2011;22:111-119.

[3] Reibnegger G. Pteridines 2015;26:135–142.

Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase 1

Romani R, Pirisinu I, Calvitti M, Pallotta MT, Gargaro M, Bistoni G, Vacca C, Di Michele A, Orabona C, Rosati J, Pirro M, Giovagnoli S, Matino D, Prontera P, Rosi G, Grohmann U, Talesa VN, Donti E, Puccetti P, Fallarino F

Department of Experimental Medicine, Department of Physics and Geology, Department of Medicine, Department of Pharmaceutical Sciences, and Department of Surgery and Biomedical Sciences, University of Perugia, Perugia, Italy; and Department of Surgery, ‘La Sapienza’ University, and iPS-Cellular Reprogramming Unit, Fondazione Casa Sollievo della Sofferenza, Mendel, Rome, Italy; and Plastic Surgery Unit, Hospital Universitario de la Ribera, Valencia, Spain

(fllfnc@tin.it)

Although human amniotic fluid does contain different populations of fetal-derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in-vitro expanded, amniotic-fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second-trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in-vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon-γ (IFN-γ), including induction of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN-γ–treated fHASCs caused significantly decreased T-cell proliferation and increased frequency in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells. Both effects required an intact IDO1 function and were cell contact-independent. An unprecedented finding in our study was that purified vesicles from IFN-γ–treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC-like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4⁺ CD25⁺ Foxp3⁺ T cells in graft-draining lymph nodes from recipient mice. Thus, fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.

Alkylglycerol monooxygenase in differentiation of 3T3-L1 fibroblasts

Sailer S, Geley S, Hermetter A, Golderer G, Werner-Felmayer G, Werner ER, Watschinger K

Division of Biological Chemistry, and Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria; and Institute of Biochemistry, Graz University of Technology, Graz, Austria

(sabrina.sailer@i-med.ac.at)

Alkylglycerol monooxygenase (AGMO) is one of eight known tetrahydrobiopterin-dependent enzymes and cleaves the ether bond of alkylglycerols and lysoalkylglycerophospholipids in a mixed function oxygenase reaction [1]. Its expression and activity can be detected in a variety of tissues and cell lines including adipose tissue. Recently we could show that AGMO is active in the murine pre-adipocyte cell line 3T3-L1 by HPLC based on the conversion of 1-O-pyrenedecyl sn-glycerol to 1-pyrenedecanoic acid.

We established stable 3T3-L1 pre-adipocyte cell lines with either diminished or increased AGMO expression by retroviral manipulation. During the course of differentiation to mature adipocytes we looked at gene expression of adipocyte markers (Fatty acid binding protein 4 and Peroxysome proliferator activated receptor γ) and visualized lipid droplet formation by using the neutral lipid dye Oil Red O.

Our results indicate that AGMO manipulation alters the fate of 3T3-L1 adipocyte conversion by activation of a yet unknown signaling pathway. Therefore we apply a high throughput inhibitor screening to identify potential candidates that are activated by the AGMO ether lipid pathway.

[1] Watschinger K & Werner ER. IUBMB life 2013;65:366-372.

Alkylglycerol monooxygenase in Dictyostelium discoideum

Seppi D, Golderer G, Hermetter A, Keller MA, Werner-Felmayer G, Watschinger K, Werner ER

Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Austria; Institute of Biochemistry, Graz University of Technology, Graz, Austria

(daniele.seppi@i-med.ac.at)

Alkylglycerol Monooxygenase is a key enzyme of ether lipid metabolism. It is the only enzyme known to cleave the ether bond of alkylglycerols and lyso-alkylglycerol phospholipids, including lyso-platelet activating factor. Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They are present in organisms ranging from bacteria, protozoa, fungi, higher plants to mammals including humans. This lipid class has a wide activity spectrum including immune-stimulatory, antibacterial and antifungal properties. Ether lipids can also increase permeability of the blood brain barrier thereby enabling delivery of drugs. Platelet activating factor (PAF), a well described potent inflammatory mediator is an example of an alkylglycerol phospholipid signaling molecule.

Alkylglycerol monooxygenase is regularly found only in bilaterial animals. However the slime mold Dictyostelium discoideum also expresses alkylglycerol monooxygenase activity. This organism is called a social amoeba due to cooperation of single celled amoebae in the formation of a fruiting body and spores upon starvation. This differentiation process is characterized by the occurrence of only two main cellular species, and has served as a model to unravel cellular signals such as cAMP.

The aim of this study is to investigate the role of alkylglycerol (glyceryl ether) monooxygenase in Dictyostelium discoideum. This will be done by to establishing D. discoideum clones in which the expression of alkylglycerol monooxygenase is up regulated or lacking and to create and validate a targeted LC-MS/MS method to analyze and quantify lipids from biological relevant classes such ether lipids. These techniques taken together provide us powerful tools for examining the physiological relevance of alkylglycerol monooxygenase on the life cycle, development and to perform a quantitative lipidomic study on the model organism D.discoideum.

Cause-effect relations between immune activity and sleep in a breast cancer survivor: A time-series analysis approach

Singer M, Peterlini S, Fritzsche K, Burbaum C, Bliem HR, Geser W, Fuchs D, Schubert C

Psychoneuroimmunology Lab, Clinic for Medical Psychology, Medical University, Innsbruck, Austria

(magdalena.singer@i-med.ac.at)

The present study on a breast cancer survivor with cancer-related fatigue (CaRF) and depression investigated the bidirectional cause-effect relations between cellular immune activity and subjective sleep variables under as ecologically valid as possible research conditions. The 49-year-old patient (breast cancer diagnosis 5 years ago, currently in remission) collected her entire urine for 28 days in 12-h intervals (from 8 p.m. to 8 a.m. and from 8 a.m. to 8 p.m.). For the purpose of this study, we used night time urine samples only resulting in time series of 28 measurements. Using these samples, urinary neopterin (cellular immune activation marker) and creatinine levels were determined using high-performance liquid chromatography (HPLC) technique. Every morning, the patient provided information on the following sleep variables: sleep quality (SQ), sleep recreational value (SRV), total sleep time (TST), total wake time (TWT), sleep latency (SL), wake after sleep onset (WASO) and awakenings during sleep period (ADS). Time series were pre-whitened by Centered Moving Average Smoothing. Cross-correlational analyses shows that increases in the positively coded sleep variables SQ and SRV were followed by urinary neopterin concentration decreases after temporal delays of 96–120 h (SQ, lag 4: r = -0.411; p = 0.044; SRV: lag 4: r = -0.472; p = 0.021) and 120–144 h (SRV, lag 5: r = -0.464; p = 0.026). Increases in the negatively coded sleep variables TWT and WASO, on the other hand, significantly preceded increases in urinary neopterin concentrations 72–96 h (TWT, lag 3: r = 0.522; p = 0.009) and 96-120 h (WASO, lag 4: r = 0.434; p = 0.033) later. No systematic effects in the other direction of effects, i.e. from urinary neopterin to sleep, were observed in this study. These results demonstrate that positive and negative sleep characteristics differentially affect cellular immunity in a breast cancer survivor with temporal delays far exceeding time intervals known from conventional group studies. This study highlights the fundamental benefit of carefully investigating directions of effects and temporal delays when studying the dynamic relationship between sleep and immune variables in cancer survivors. Should these findings be replicated in further studies, they would have the potential to improve the clinical management of sleep-disturbances in breast cancer survivors.

Activated immune system and inflammation markers in pediatric patients with seasonal allergic conjunctivitis

Sipahi H, Cinici E, Saziye Palabiyik SS, Baydar T

Department of Toxicology, Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey; Department of Ophthalmology, Regional Training and Research Hospital, and Department of Toxicology, Faculty of Pharmacy, Atatürk University, Erzurum, Turkey; and Department of Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey

(handesipahi@hotmail.com)

Allergic conjunctivitis is one of the most common syndrome in ocular disorders with a prevalence ranging from 5-22% in the general population. Th2 type cytokines have a pathogenic role in ocular allergic diseases but inflammatory cytokines like the typical Th1-cytokine, IFN-γ, is also over-expressed during the active inflammatory phase of the ocular allergic diseases. Neopterin production is induced by IFN-γ which may also change during allergic inflammation. IFN-γ also stimulated the enzyme indoleamine 2,3-dioxygenase (IDO) that converts the essential amino acid tryptophan to kynurenine. The aim of this study was to evaluate the serum neopterin and nitrite levels, kynurenine to tryptophan ratio (Kyn/Trp) as indicator of IDO activation in 44 pediatric patients with seasonal allergic conjunctivitis, along with these values in 33 healthy pediatric subjects and to assess the correlation of these biomarkers related with inflammatory response in seasonal allergic conjunctivitis. According to our results, serum neopterin levels were significantly increased in patients compared with the controls (p <0.05) and neopterin levels were significantly correlated with symptom score (rs=0.317, p <0.05) and Kyn/Trp ratio (rs=0.448, p <0.05). Serum nitrite levels and Kyn/Trp ratio did not differ between the groups. Tryptophan levels were higher in healthy controls compared with patients (p <0.05). In conclusion, increased neopterin levels due to Th1 type immune system activation might be serve as a biomarker in allergic conjunctivitis but it should be evaluated with further studies in a large study group of different allergic disorders.

*The study was supported by the Scientific Research Council of Atatürk University (number 2014/169)

[1] Kositz C, et al. Int Arch Allergy Immunol 2008;147:35-40.

[2] Leonardi A, et al. Exp Eye Res 2013;117:106-117.

HIV and the central nervous system: not just neuroinflammation

Tavano B, Tsipouri V, Keegan MR, Royle CM, Ferguson D, Almond N, Boasso A

Centre for Immunology & Vaccinology, Chelsea & Westminster Hospital, Imperial College London, UK; Royal Brompton & Harefield NHS Foundation Trust, London, UK; ViiV Healthcare Ltd., Brentford, Middlesex, UK; National Institute for Biological Standards and Controls (NIBSC) – Potters Bar, UK

(a.boasso@imperial.ac.uk)

Human immunodeficiency virus (HIV)-associated dementia (HAD) developed during late stage HIV diseases in 20–30% of patients before the introduction of antiretroviral therapy (ART) [1]. ART suppresses HIV replication to undetectable levels in most patients, and has virtually eliminated severe neurological complications. However, up to 40% of HIV-infected individuals under ART still develop HIV-associated cognitive impairment that can affect daily life and disrupt adherence to ART [2]. HIV-induced neuroinflammation may be present despite very low levels of viral replication within the central nervous system (CNS) [3]. Neurologically healthy HIV-infected patients receiving effective ART showed widespread microglial activation, as measured by binding of the transporter protein (TSPO) radioligand [¹¹C]PBR28 detected by PET [4]. Experimental infection of macaques with the simian immunodeficiency virus (SIV) is a reliable model for HIV disease, and neuroinflammation is observed in neurologically healthy SIV-infected macaques with undetectable viraemia [5].

We tested the expression of 84 genes encoding for inflammatory mediators (chemokines, cytokines and their receptors) in biopsies collected post-mortem from the cerebrum (frontal lobe), midbrain and cerebellum of SIV-negative (SIV-; N=3) and SIV-positive (SIV+; N=9) macaques. Significantly increased expression of 3/84 genes, was observed in the midbrain of SIV+ animals. Conversely, 12/84 tested genes were significantly down-regulated in the cerebrum of SIV+ compared to SIV- animals. No alterations of inflammatory genes expression was observed in the cerebellum. Interestingly, SIV+ macaques which had undetectable viraemia at the time of euthanasia (N=2/9) showed the same gene expression pattern as SIV+ macaques with high viraemia (N=7/9). Gene ontology and pathway analysis suggested that alterations of the pathways regulated by interferon (IFN)-γ or interleukin (IL)-6 were predominant in both midbrain and cerebrum. Minor alterations were observed in an array of genes associated with neurotoxicity.

Our data collectively suggest that, although microglia activation may be widespread in the CNS during HIV/SIV infection, the immunological outcome may differ in different anatomical regions. Further studies are necessary to determine the implications of these anatomical differences for both neurological disease and for the perpetuation of a reservoir of HIV/SIV-infected cells in the CNS as an immune privileged site.

[1] Gonzalez-Scarano F & Marin-Garcia J. Nat Rev Immunol 2005; 5: 69-81.

[2] Antinori A, et al. Neurology 2007; 69: 1789-99.

[3] Valcour V, et al. Curr HIV/AIDS Rep 2011; 8: 54-61.

[4] Vera JH, et al. Neurology 2016; ePub ahead of print.

[5] Clarke S, et al. J Neurovirol 2012; 18: 100–112.

Iron and the route to mitochondria

Volani C, Haschka D, Demetz E, Doerrier C, Gnaiger E, Weiss G

Department of Internal Medicine VI, Medical University of Innsbruck; andOroboros Instruments, Innsbruck, Austria

(chiara.volani@i-med.ac.at)

Iron is a fundamental co-factor for several cell processes, including oxidative phosphorylation, and mitochondria are the major sites of iron-utilization. Besides being central part of mitochondrial complex I-IV enzymes of the electron transport system, iron also regulates citric acid cycle activity by modulating mitochondrial aconitase expression. As a result, mitochondrial iron accumulation is tightly regulated thanks to the presence of mitochondrial iron transporters, namely mitoferrin 1 and mitoferrin 2. However, so far, little information is available on how mitochondrial function is affected by functional and genetic alterations of iron homeostasis, and how to best measure tissue mitochondrial activity and its interaction with iron homeostasis in vivo; therefore we aimed at investigating the relevance of mitochondrial iron loading and the underlying mechanisms.

To access the impact of iron on mitochondrial respiration we studied mitochondrial respiration in livers of 10-week old FVB mice, receiving either normal- or high iron-diet (25 g/kg) two weeks before being sacrificed. The liver was collected and stored in Custadiol prior to homogenization in MiR05. Mitochondrial leak respiration, complex I and II maximal oxidative phosphorylation together with non-coupled respiration of the homogenates were assessed by means of high resolution respirometry (OROBOROS Instruments, Austria).

Our ongoing experiments indicate that mitochondrial function testing can be successfully performed in mouse tissues. Analyses of liver samples indicate that dietary iron supplementation triggers changes in oxidative phosphorylation, and has a direct impact on the activity of the electron transfer system complexes.

The use of high-resolution respirometry (OROBOROS Instruments, Austria) represents a powerful and reliable tool to investigate mitochondrial respiration, and possibly provide information on the tissue mitochondrial activity.

Alkylglycerol monooxygenase physiology-what is known so far

Watschinger K, Keller MA, Golderer G, Werner-Felmayer G, Werner ER

Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

(katrin.watschinger@i-med.ac.at)

Tetrahydrobiopterin-dependent alkylglycerol monooxygenase was first described in 1964. In 2010, we assigned its sequence to former transmembrane protein 195. The physiological role of alkylglycerol monooxygenase however remains so far largely unknown.

Here we present an update on the anabolic and metabolic routes of ether lipids, including the free alkylglycerols which are the natural alkylglycerol monooxygenase substrates and present important members of this lipid class. We show that alkylglycerol monooxygenase is regulated in primary macrophages when stimulated with inflammatory and anti-inflammatory cytokines and knockdown of the enzyme in a macrophage cell line interferes with endogenous levels of certain lipids including the free alkylglycerols which points to an important role of this enzyme in lipid homeostasis of the cell. Work in C. elegans showed that the cuticle sensitivity of tetrahydrobiopterin deficient worms is conferred by impact on alkylglycerol monooxygenase.

There are still some open questions in the field of tetrahydrobiopterin or alkylglycerol monooxygenase metabolism, e.g. embryonic lethality of mice deficient in the key enzyme of tetrahydrobiopterin biosynthesis. Relevant issues are presented together with a working hypothesis of how alkylglycerol monooxygenase could be responsible for these so far unexplained effects.

Type-1 versus type-2 epithelial differentiation determines IDO and AhR regulation: novel insights in allergy

Zissler UM, Chaker AM, Effner R, Ulrich M, Guerth F, Piontek G, Dietz K, Regn M, Knapp B, Theis FJ, Schmidt-Weber CB

Center of Allergy & Environment (ZAUM), Technical University of Munich and Helmholtz Center Munich, German Research Center for Environmental Health, Germany, Member of the German Center for Lung Research (DZL); Department of Otorhinolaryngology and Head and Neck Surgery, Medical School, and Institute of Pharmacology and Toxicology, Medical School, Technical University of Munich; Institute of Computational Biology, Technical University of Munich and Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. Department of Mathematics, Technical University of Munich, Garching, Germany; Division of Innate Immunity, Research Center Borstel, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany

(csweber@tum.de)

Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are lead cytokines for the differentiation of T helper type 1 and 2 (Th1 and Th2) cells. Both cytokines induce fate-decisive transcription factors such as GATA3 and TBX21 that antagonize the polarized development of opposite phenotypes by direct regulation of each other’s expression along with many other target genes. Although it is well established that mesenchymal cells directly respond to Th1 and Th2 cytokines, the nature of antagonistic differentiation programs in airway epithelial cells is only partially understood.

In this study, primary normal human bronchial epithelial cells (NHBEs) were exposed to IL-4, IFN-γ, or both and genome-wide transcriptome analysis was performed. The study uncovers an antagonistic regulation pattern of IL-4 and IFN-γ in NHBEs, translating the Th1/Th2 antagonism directly in epithelial gene regulation. IL-4- and IFN-γ-induced transcription factor hubs form clusters that include the aryl hydrocarbon receptor (AhR), present in antagonistically and polarized gene regulation networks. Furthermore, indoleamine 2,3-dioxygenase (IDO) is induced by IFN-γ while down-regulated by IL-4. This study shows that the impact of environmental metabolites such as tryptophan and its products will be differentially received depending of the immunological make-up of the exposed epithelial cells.