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Official Journal of the International Society of Pteridinology

Editor-in-Chief: Fuchs, Dietmar

1 Issue per year

IMPACT FACTOR 2017: 0.586

CiteScore 2017: 0.53

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Volume 26, Issue 3


34th International Winter Workshop / Clinical, Chemical and Biochemical Aspects of Pteridines and Related Topics

Published Online: 2015-06-20 | DOI: https://doi.org/10.1515/pterid-2015-0007

These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax, and usage are the authors’ responsibility.

Society for Exploitation of Education and Research in Immunology and Infectious

Diseases, Innsbruck, Austria

in collaboration with

The International Society of Pteridinology


The Austrian Society of Laboratory Medicine and Clinical Chemistry

Held in Innsbruck, Tyrol, Austria,

February 24th–27th, 2015

Scientific committee: Dietmar Fuchs (Innsbruck), Andrea Griesmacher (Innsbruck), Bohuslav Melichar (Olomouc), Gilbert Reibnegger (Graz), Barbara Strasser (Hall), Guenter Weiss (Innsbruck) and Ernst R. Werner (Innsbruck)

Organization: Dietmar Fuchs, Sektion für Biologische Chemie, Biozentrum, Medizinische Universität Innsbruck, Innrain 80, 6020 Innsbruck, Austria, e-mail:

Circulating neopterin and citrulline concentrations in patients with germ-cell tumors during chemotherapy

Bartoušková M, Študentová H, Pejpková I, Zezulová M, Adam T, Melichar B

Palacký University Medical School and Teaching Hospital, Olomouc, Czech Republic

( )

Germ-cell tumors are relatively rare neoplasms that affect mostly young adults. The poor prognosis of these tumors has significantly improved after the advent of platinum compound, and most patients with metastatic germ cell tumors can be currently cured with combined modality treatment that comprises surgery, chemotherapy or radiotherapy. With the improved long-term prognosis, acute and chronic side effects of treatment represent an important issue. In the present study, we evaluated serum neopterin and plasma citrulline, a biomarker of gut damage in germ-cell tumor patients before and during 3 to 4 cycles of standard platinum-based chemotherapy. We report here the pilot results in the first 15 patients. Baseline serum neopterin concentrations were increased in a substantial proportion of patients, while plasma citrulline was mostly within the normal range. A significant negative correlation was observed between baseline serum neopterin and hemoglobin concentrations while positive concentration was noted between peripheral blood leukocyte count and neopterin. Changes in the investigated parameters were noted during the course of therapy. The study is ongoing.

Growing impact of psychoimmunological findings to understand pathogenesis of severe mental illness

Bechter K, Maxeiner H, Kühne L, Reiber H, Fuchs D, Schneider M

Clinic for Psychiatry and Psychotherapy II, Ulm University, Germany; Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

( )

Etiology and pathogenesis of severe mental illness, core is the affective and schizophrenic spectrum, remains widely unknown, though many researchers assume some biological basis. CSF analysis is a key bodily fluid to assess rather directly the pathogenetic processes involved in brain diseases. We demonstrated in several research projects, focused on therapy resistant cases of severe mental illness, in about 40 % of patients some CSF abnormalities by conventional sophisticated CSF analysis, in addition increased CSF neopterin in about 30 %, together indicating immune pathological responses within the intrathecal space in more than 50 % of cases. Also blood CSF barrier dysfunction was found summing up to 70 % of cases with CSF pathology. However in 100 % of these cases we demonstrated CSF cytokine abnormalities, often paralleled by corresponding though not identical cytokine abnormalities in blood. Together our results reveal immune pathology in a large subgroup of patients of the affective and schizophrenic spectrum, focused within the intrathecal spaces.

Influence of carbon nanotubes, ZnO and gold-doped TiO2 nanoparticles on human PBMC in vitro

Becker K, Herlin N, Bouhadoun S, Gostner JM, Ueberall F, Schennach H, Fuchs D

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

( )

Nanomaterials are increasingly produced and used throughout recent years. Consequently the probability of exposure of humans to nanoparticles has drastically risen. 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 carbon nanotubes (CNT), zinc oxide (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). PBMC were stimulated with mitogen phytohaemagglutinin (PHA). Activated T-cells release pro-inflammatory cytokines, leading to an enhanced formation of neopterin and tryptophan breakdown. Furthermore myelomonocytic THP-1-blue cells, a reporter-cell line, can detect the activation of the redox-sensitive transcription factor nuclear factor-κB (NF-κB) [3]. Stimulation of THP-1-blue cells with lipopolysaccharide (LPS) resulted in the activation of NF-κB, which could be measured by assessing the SEAP activity in a colorimetric assay (n = 4). 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 (37.5 – 150 μg/ml) resulted in a decrease of neopterin levels. The same observations were found for kynurenine to tryptophan ratios (Kyn/Trp) representing tryptophan breakdown rates. In THP-1-blue cells CNT were able to raise NF-kB activation significantly in LPS-stimulated cells. TiO2 and gold doped TiO2 (9.37 – 150 μg/ml) were able to increase neopterin production in both stimulated and unstimulated PBMC. However, Kyn/Trp levels were only influenced in stimulated cells, where inhibition of tryptophan breakdown could be observed. Also ZnO treatment resulted in distinct effects. Lower concentrations (2.34-18.75 μg/ml) acted 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. Furthermore results obtained with ZnO (50nm and 100nm and bulk material) were not size dependent.

Our results show that not all types of nanoparticles had similar effects on PBMC and THP-1-blue cells in vitro. Every type showed particular results and have to be explored in more detail on other cell types. Additionally exposure to nanoparticle during inflammatory conditions may induce different effects as compared with a “healthy” and unstimulated milieu.

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.

IDO and related pathways as targets for HIV immunotherapy

Boasso A

Centre for Immunology and Vaccinology, Chelsea & Westminster Hosp., Imperial College London, UK

( )

Increased Tryptophan (Trp) catabolism via the Kynurenine (Kyn) pathway is observed in several chronic inflammatory and infectious diseases, and is caused by increased indoleamine 2,3-dioxygenase (IDO) activity. In the particular case of HIV-1 infection, IDO dysregulation is considered to contribute to different aspects of immunopathogenesis, including immune deficiency, neurological impairment and tissue fibrosis. Increased IDO activity may result from direct activation of certain immune cell subsets by HIV (eg. plasmacytoid dendritic cell; pDC), or may be driven by the generalized immune activation observed during chronic HIV infection via inflammatory cytokines (such as type I and type II interferons) and ligand-receptor interactions (eg. binding of cytotoxic T lymphocyte antigen (CTLA)-4 to its ligands B7.1 and B7.2). Immunotherapeutic approaches which target IDO directly or indirectly are being considered for the treatment of HIV infection and have been tested mainly at preclinical level. The competitive IDO inhibitor 1-methyl-D-tryptophan (D-1mT) has been tested in both humanized mice and SIV/macaques with encouraging results, indicating that IDO inhibition may favour immune-mediated control of HIV replication [1,2]. Strategies interfering with the binding of CTLA-4 to its ligands B7.1 and B7.2 have shown ambiguous results, but the effect of CTLA-4 blockade may only be partially dependent on IDO suppression [3,4]. In addition, simultaneous administration of D-1mT and anti-CTLA-4 to Rhesus macaques caused fatal pancreatitis, calling for caution when considering strategies which may reverse immune tolerance [5]. Other therapeutic options aim at dampening the generalised immune activation by using compounds with broad anti-inflammatory properties, particularly those interfering with pDC activation, a notable example is that of the antimalaric drug chloroquine and its derivative hydroxychloroquine (CQ and hCQ). However, the direct effect of these compounds on IDO activity in vivo is yet to be determined. Thus, inhibition of IDO or IDO-related pathways may represent a valid therapeutic option in the setting of HIV infection, but its application is still limited by the paucity of specifically designed comparative preclinical and clinical studies.

[1] Potula R, et al. Blood 2005; 106(7): 2382-90.

[2] Boasso A, et al. J Immunol 2009; 182: 4313-20.

[3] Hryniewicz A, et al. Blood 2006; 108: 3834-42.

[4] Cecchinato V, et al. J Immunol 2008; 180: 5439-47.

[5] Vaccari M, et al. J Virol 2012; 86: 108-13.

Interferon-γ-mediated immune activation in patients with severe symptomatic aortic stenosis and implications of baseline inflammatory markers for risk stratification in patients undergoing transcatheter aortic valve implantation

Csordas A, Fuchs D, Reibnegger G, Nietlispach F, Maier W, Stähli BE, Binder RK, Cahenzli M, Lüscher TF

Department of Cardiology, University Heart Center Zürich, Zürich, Switzerland; Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; and Institute of Physiological Chemistry, Center of Physiological Chemistry, Medical University, Graz, Austria

( )

Transcatheter aortic valve implantation (TAVI) has emerged as a viable treatment option for patients with severe symptomatic aortic stenosis and a high or prohibitive surgical risk. Neopterin, a pteridine mainly synthesized by activated macrophages, is considered a stable integral of TH1 immune activation, and elevated neopterin levels have a solid track record as risk estimates for mortality in atherosclerosis-related diseases. We prospectively included 128 patients undergoing TAVI. Neopterin, kynurenine, tryptophane, tyrosine and phenylalanine levels were measured at baseline as well as at postoperative day 1-3. Neopterin and the Euroscore II were dichotomized at the upper percentile, and paramteric Royston-Parmar proportional odds models were employed relating biomarkers and clinical risk scores to non-procedural mortality. Mortality was higher for patients with elevated neopterin (>21 nmol/L, upper percentile) (18.18% vs. 5.05%, P = 0.018) and Euroscore II (> 7.5, upper percentile) (18.75% vs. 6.1%, P = 0.031). In bivariate analysis adjusted for Euroscore II, elevated neopterin (>21 nmol/L) persisted as an independent predictor of mortality (HR 2.41 95% CI [0.99-13.6], P = 0.050). A similar finding was obtained in an external confirmation cohort encompassing 207 patients. In conclusion, systemic markers of increased immune activation, as evidenced by enhanced tryptophan degradation and neopterin production, provide an incremental risk estimate for mortality beyond canonical surgical risk scores. Our data support a role for a biomarker-guided strategy for sharpening discrimination of high-risk patients in advance of TAVI and underscore the role of baseline inflammation for prognosis in patients with severe aortic stenosis.

Specific in-vivo requirements for tetrahydrobiopterin revealed by cell-specific deletion of GTP cyclohydrolase I

Channon KM

Cardiovascular Medicine, R&D and NIHR Biomedical Research Centre, John Radcliffe Hospital University of Oxford, Oxford, Great Britain ( )

Abstract not received in time.

HFE deficiency critically affects cholesterol homeostasis in mice

Demetz E, Haschka D, Heim C, Auer K, Aßhoff M, Schroll A, Dichtl S, Hilbe R, Lener D, Seifert M, Tymoszuk P, Pechlaner R, Willeit J, Kiechl S, Theurl I, Tancevski I, Weiss G

Medical University of Innsbruck, Department of Internal Medicine VI, Infectious Diseases, Immunology, Innsbruck, Austria

( )

Iron metabolism plays a crucial role in diseases characterized by chronic inflammation including atherosclerosis. Specifically, iron overload has been linked to an increased risk for atherosclerosis development, and with its sequels stroke and myocardial infarction. However, the underlying mechanism has not been elucidated thus far. To study this we used apoE-/- mice as a model of dyslipidemia which develop spontaneous atherosclerosis over time and crossed these with Hfe-/- mice, a model of genetic iron overload. When modulating dietary iron supplementation, we did not observe any differences in cholesterol metabolism in both wildtype and apoE-/- mice. However, mice lacking Hfe, different iron loading markedly affected plasma cholesterol levels. In both studied models, i.e. wildtype and apoE-/- mice, high iron feeding resulted in a strong induction of hepatic LDL receptor which was associated with decreased plasma LDL cholesterol levels. Furthermore, high iron fed animals displayed enhanced serum cholesterol efflux capacity, which may additionally inhibit cholesterol deposition in the arterial wall. Both, enhanced LDL cholesterol clearance together with enhanced macrophage cholesterol efflux may be responsible for the significant reduction in atherosclerotic lesion area observed in high-iron fed Hfe-/- apoE-/- mice. In summary, we show that disruption of the gene coding for Hfe affects cholesterol metabolism and atherogenesis, presumably through regulation of hepatic LDL receptor and by modulating cholesterol efflux capacity, both mechanisms shown to be significantly influenced by dietary iron supplementation.

Development of an ELISA for neopterin measurement

Dressler D

F&E Gastroenterology and Lateral Flow Development, R-Biopharm AG, Darmstadt, Germany

( )

An enzyme linked immunoassay for the detection of neopterin from human serum was developed. Results of 48 human serum samples were compared with a commercial ELISA from BRAHMS, Hennigsdorf, Germany. The new test revealed very good sensitivity and there existed a significant correlation between the obtained results with both assays, offering the possibility to distinguish serum samples from sick or healthy subjects.

Serum leptin levels are associated with the interferon gamma +847T/A polymorphism (rs62559044)

Egger C, Wallner V, Weghuber D, Zelzer S, Paulweber B, Fuchs D, Mangge H, Baumgartner GB

Clinical Institute of Internal Medicine, and Department of Pediatrics, Paracelsus Medical University, Salzburg, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University, Graz, Austria, Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

STYJOBS/EDECTA (details: http://clinicaltrials.gov/ct2/show/NCT00482924)

( )

Leptin has initially been described as hormone that is released by adipose tissue and controls nutritional intake by binding and activating POMC neurons in the hypothalamus. However, leptin has pleiotropic functions and has been shown to have pro-inflammatory and immunomodulatory effects. In combination with interferon gamma (IFNγ), leptin stimulates TH1 T-cells, which cause inflammation promoting insulin resistance. There is a clear established connection between serum leptin levels and IFNγ levels, as ectopic leptin stimulates IFNγ release. Santucci et al. (2011) found that plasma leptin levels are positively correlated to the basal amount of IFNγ present at 24 h in culture supernatants from tuberculosis patients [1]. Hence, activation of IFNγ might lead to altered leptin levels in serum.

IFNγ is a potent inflammatory mediator that carries a polymorphism (+847T/A; rs62559044) in the first intron which is located within a canonical binding site of the activating transcription factor NF-κB and its functional importance was shown as the T-allele gives rise to increased levels of IFNγ. We developed a simple genotyping method for this polymorphism and genotyped the IFNγ +874 A/T polymorphism in 508 patients (60% females) of the STYJOBS/EDECTA cohort, which has been set up to investigate the preclinical phase of obesity by a well defined cohort of young and middle aged overweight/obese and normal weight subjects [2].

In a general linear model, rs 62559044 associated significantly with leptin levels (p= 0.032). The effect is sex specific, as the association between rs 62559044 and serum leptin levels in women is highly significant (p=0.003) while we detected no significant association in males. The effect is allele specific: using the Mann-Whitney Test, the presence of the A allele versus the TT genotype did not return significant differences of serum leptin levels depending on IFNγ allelic status, while comparing the presence of the T allele versus the AA genotype resulted in a significant association (p=0.004). Therefore, we conclude that the T-allele of rs62559044 might increase serum leptin levels.

[1] Santucci N, et al., PLOS One 2011;6(10):e26363.

[2] Mangge H et al., Obesity (Silver Spring) 2013;21:E71-7.

Volatile breath markers of lung infection in mechanically ventilated patients

Filipiak W, Sponring A, Filipiak A, Ager C, Nagl M, Beer R, Troppmair J, Amann A†

Breath Research Institute of the Austrian Academy of Sciences, Dornbirn, Austria; Department of Anaesthesiology and Critical Care Medicine, Department of Hygiene, Microbiology and Social Medicine, Neurological Intensive Care Unit, Department of Neurology, andDaniel-Swarovski Research Laboratory, Department of Visceral-, Transplant- and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria

( )

†dedicated to the memory of Prof Anton Amann who passed away January 6, 2015

The routinely used microbiological diagnosis of Ventilator-Associated Pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Thus, the most desirable improvement related to diagnosis of pneumonia is an earlier and non-invasive detection of the outbreak of infection and faster identification of the pathogens. This might be achieved by monitoring the presence of pathogen-derived volatile metabolites in the alveolar air of mechanically ventilated patients. For this purpose, in vitro experiments with microorganisms frequently found in VAP patients, i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia, Haemophilus influenzae and Candida albicans, were performed to identify pathogen-specific volatile organic compounds (VOCs).

Numerous VOCs were released by investigated bacteria and fungi species in a concentration range varying from pptv to ppmv level. The clear differences in the bacteria-specific profiles of VOCs production were found, particularly hydrocarbons released by Streptococcus pneumonia and Haemophilus influenzae and for aldehydes which were taken up by P. aeruginosa but released by S. aureus and for. Importantly, the in vitro experiments were performed under culture conditions and at bacterial densities which relate to the in vivo situation in the lungs of VAP patients. Moreover, the significant VOCs release was found already 1.5 to 3 hours after inoculation, and their concentration profiles were very well correlated to the proliferation rate of cultured microorganisms.

In a next step a clinical study at the Neurological Intensive Care Unit was performed to clarify whether (i) the volatile metabolites produced by pathogens in vitro can be detected in alveolar air of mechanically ventilated patients, and (ii) changes in bacterial metabolites reflect disease progression and therapeutic interference. The results of breath analysis were referred to the patient’s health status, in particular to the level of C-reactive protein (CRP). Pneumonia was diagnosed on the basis of clinical (e.g. onset of fever, CRP level), microbiological (isolates) and imaging (CT or X-Ray) data. The developed methodological platform was proven to be suitable for application in the ICU whereby end-tidal air was collected directly from the respiratory circuit of MV patients. The relationship between the concentration profiles of selected breath-VOCs and the course of the microbial lung infection was found. A change in the breath-VOC patterns was observed in relation to the patients’ recovery.

In summary, we observed characteristic VOC patterns for each microorganism that, though limitation to a small patient cohort, was suitable to detect the presence of pathogens in mechanically ventilated patients. The here presented results set fundament for further clinical study, preferably with real-time mass spectrometric techniques in ICU settings.

Neopterin concentrations and tryptophan breakdown rates in the elderly are influenced by life style and dietary habits

Fuchs D, Gostner JM, Sánchez-Flores M, Millán-Calenti JC, Marcos-Pérez D, Maseda A, Pásaro E, Strasser B, Lorenzo-López L, Laffon B, Valdiglesias V

Biocenter, Medical University, Innsbruck, and Institute for Nutritional Sciences and Physiology, UMIT, Hall, Austria; DICOMOSA Group, Department of Psychology, Area of Psychobiology, and Gerontology Research Group, Department of Medicine, Universidade da Coruña, Spain.

( )

Frailty is placed in an intermediate position between being functional and the development of functional impairment and/or comorbidity directly associated with a disease process. Numerous factors play a role in the development of frailty including immobility (decreased physical activity), balance impairment, depression and cognitive impairment. Phenotypic criteria that indicate compromised energetics include low grip strength, low energy, slowed waking speed, low physical activity, and/or unintentional weight loss [1]. Chronic immune system activation and inflammation seem to contribute to the development of frailty, e.g., enhanced neopterin concentrations have been described recently in frail individuals [2].

Smoking status and dietary habits are considered to impact on the performance. In the in vitro system of human peripheral blood mononuclear cells (PBMC), immunosuppressant properties of several antioxidant vitamins and other compounds were shown, reducing neopterin production and tryptophan breakdown rates [3]. Moreover, significant inverse correlations between higher neopterin blood levels and lower concentrations of several antioxidant compounds and vitamins were observed in patients at cardiovascular risk [4]. Likewise, smokers were found to present lower neopterin and Kyn/Trp levels than non-smokers [5-7].

In this study, in 239 individuals (35 non-frail, 118 pre-frail and 86 frail) aged >63 (mean ± SD: 79.4+8.86) years serum neopterin concentrations were determined by ELISA (BRAHMS, Germany) and kynurenine to tryptophan ratios (Kyn/Trp) by HPLC, as biomarkers of immune activation. Results were compared to dietary habits including intakes of coffee, tea and alcohol as well as to smoking status. Intake of coffee, tea and alcohol was associated with lower neopterin and Kyn/Trp concentrations, whereas tryptophan was higher. This was especially true in those who drank alcoholic beverages. Neopterin and Kyn/Trp concentrations were lower and tryptophan levels higher in smokers vs. non-smokers. Tobacco consumers presented significantly higher blood tryptophan levels. No significant associations existed between the number of daily cigarettes and the number of years smoked and the concentrations of biomarkers.

The association between immune system activation and the disturbed metabolism of tryptophan and neopterin in frail individuals appears to be related to smoking habits and nutritional aspects. Intake of nutrients rich in antioxidants like tea and coffee as well as smoking may influence immunoregulatory pathways. In accordance with in vitro data, antioxidant contents of beverages could be of importance to underlie this effect, e.g., vitamins, resveratrol and humulones in beverages, but also carbon monoxide in smokers could explain our findings. In a similar way, polyphenols from beer were found to reduce inflammatory biomarkers related to atherosclerosis [8]. Data of our study may point to a role of such behavior in suppressing the activated immune system in older age. Thereby, the food compounds could also influence nutrition behavior when intake of tryptophan-rescuing food contributes to mood enhancement. The metabolic alterations especially of tryptophan could be important for the amelioration of specific symptoms like depression and cognitive deficits in the frail elderly. Moreover, higher serum tryptophan levels were observed to relate to lower mortality in cardiovascular disease [9].

[1] Fried LP, et al. J Gerontol A Biol Sci Med Sci 2001;56:M146–56.

[2] Leng XS, et al. Age Ageing 2011;40:475–81.

[3] Gostner J, et al. Curr Pharm Des 2014;20:840-9.

[4]Murr C, et al. Atherosclerosis 2009;202:543-9.

[5] Diamondstone LS, et al. J Clin Immunol 1994;14:368-74.

[6] Walter RB, et al. Clin Chem Lab Med 2003;41:1314-9.

[7] Pedersen ER, et al. Arterioscler Thromb Vasc Biol 2011;31:698-704.

[8] Chiva-Blanch G, et al. Nutr Metab Cardiovasc Dis 2015;25:36-45.

[9] Murr C, et al. Eur J Clin Invest 2015 (in press).

Freezing conformational dynamics in phenylalanine hydroxylase with pharmacological chaperones

Fuchs JE, Santos-Sierra S, Lagler FB, Kirchmair J

Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, United Kingdom; Molecular and Clinical Pharmacology, Innsbruck Medical University, Austria; Institute for Inborn Errors of Metabolism, Paracelsus Medical University, Salzburg, Austria; Center for Bioinformatics, University of Hamburg, Germany

( )

Inherited mutations may lead to protein misfolding and thus loss of function. Small molecule pharmacological chaperones aim to rescue proteins from misfolding by stabilization of the fold [1]. Dysfunction of phenylalanine hydroxylase causes the well-studied genetic disease phenylketonuria, thus turning pharmacological chaperones stabilizing this enzyme against degradation into promising drug candidates [2]. In an attempt to rationalize chaperone activity on atomistic level, we perform molecular dynamics simulations to characterize intrinsic flexibility of phenylalanine hydroxylase, an enzyme with a complex energy hypersurface and several regulatory pathways [3,4,5]. We show that conformational dynamics of the protein are frozen considerably in presence of its co-factor tetrahydrobiopterin as well as pharmacological chaperone candidates. The observed reduction of the enzyme’s conformational entropy [6] correlates to the experimentally observed stabilization versus proteolytic degradation, thus showing the potential of the simulation approach to guide development of innovative fold stabilizing agents.

[1] Santos-Sierra S, et al. Human Molecular Genetics 2012;21:1877-87.

[2] Underhaug J, et al. Curr Top Med Chem 2012, 12, 2534-5.

[3] Fuchs JE, et al. PLoS One 2012;7:e53005.

[4] Ronau JA, et al. Eur Biophys J 2013;42:691-708.

[5] Fuchs JE, et al. Pteridines 2014;25:33-39.

[6] Huber RG, et al. Protein Sci 2015;24:174-81.

Determination of phenylalanine, tyrosine, tryptophan and kynurenine in plasma of rat model for tauopathies by HPLC with fluorescent and mass spectrometry detection

Galba J, Michalicova A, Parrak V, Novak M, Kovac A

Axon Neuroscience SE and Faculty of Pharmacy Commenius University, Bratislava, Slovakia

( )

We developed and validated a simple and sensitive ultra-performance liquid chromatography (UPLC) method for the analysis of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm × 50 mm, 1.7 μm particle size) using a 4 minutes ammonium acetate (pH 5) gradient and detected by Fluorescence and positive ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. The procedure was validated in compliance with the FDA guideline [1]. The limits of quantification (LOQ) were 0.125 μmol/L for Kyn and 1.25 μmol/L for Phe, Tyr, Trp. (calculated from calibration curves). The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was in the range of 95.6-108%. The inter-day precision (n = 5 days), expressed as % RSD, was in the range 5-12%. The benefit of using UPLC is a short analysis period and thus, a very good sample throughput. Using this method, we analysed plasma samples from transgenic rat model for tauopathies.

[1] US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Method Validation, 2001. http://www/fda.gov/cder/guidance/index.htm.

Effect of occupational mercury exposure on neopterin profile and tryptophan degradation in dental technicians

Girgin G, Tutkun E, Ünüvar S, Palabiyik SS, Yilmaz OH, Baydar T

Department of Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara; Ankara Occupational Diseases Hospital, Ankara; Department of Toxicology, Faculty of Pharmacy, Inönü University, Malatya; Department of Toxicology, Faculty of Pharmacy, Atatürk University, Erzurum; Turkey

( )

Amalgam has been widely used as a restorative dental material for over 150 years. Most standard dental amalgam formulations contain approximately 50% elemental mercury in a mixture of copper, tin, silver, and zinc. Mercury is a highly volatile metal, can easily vaporize to a colorless and odorless gas. It has been demonstrated that mercury is released from dental amalgam which is increased by chewing, eating, brushing and drinking hot liquids. Beside this, mercury is the main occupational exposure source for dental workers during amalgam preparation. It is known that mercury exposure causes immune modulation in humans. In this study, it was aimed to evaluate the changes in neopterin levels and tryptophan degradation in dental technicians. 135 dental technicians who applied for routine check-up to the Occupational Diseases Hospital were recruited the study. Spot urinary mercury concentrations were detected by ICP-MS. Serum neopterin levels were determined by ELISA while tryptophan and kynurenine levels were analyzed by HPLC. The results were compared to 165 healthy controls. It was observed that occupational mercury exposure had an apparent effect on neopterin levels. The significant decrease in neopterin levels and kynurenine/tryptophan in dental technicians compared to healthy controls (both, p <0.01) indicates a possible immune suppression with occupational mercury exposure. Moreover, urinary mercury concentrations and serum neopterin levels had a significant positive correlation (p <0.05), while serum kynurenine-to-tryptophan ratios and mercury levels had a negative, non-significant relationship (p>0.05). The relationship between urinary mercury levels as an indicator of occupational mercury exposure and neopterin reminded an effect on T-cell mediated immune response; however, this effect was not observed in tryptophan degradation.

Black tea consumption increases blood kynurenine concentrations - a randomized controlled trial

Gostner JM, Becker K, Croft KD, Woodman RJ, Puddey IB, Fuchs D, Hodgson JM

Divisions of Medical Biochemistry and of Biological Chemistry, Medical University of Innsbruck, Austria; School of Medicine and Pharmacology, University of Western Australia, Perth, Western Australia, Australia and Flinders Centre for Epidemiology and Biostatistics, School of Medicine, Flinders University, Adelaide, South Australia, Australia

( )

Circulating neopterin and the ratio of kynurenine to tryptophan (Kyn/Trp) concentrations are biomarkers of immune activation that have been linked to cardiovascular and total mortality [1,2]. In vitro data show that antioxidant compounds including tea flavonoids reduce immune stimulation and down-stream biochemical pathways such as neopterin formation and tryptophan breakdown in human peripheral blood mononuclear cells [3,4]. We aimed to assess the effects of regular black tea consumption on tryptophan and neopterin metabolism in vivo [5].

Participants were healthy individuals with no major illnesses, having normal to mildly elevated systolic blood pressure. They were randomly assigned to consume 3 cups per day of either powdered black tea solids (tea; n = 45) or a flavonoid-free caffeine-matched beverage (control; n = 49). Serum concentrations of tryptophan, kynurenine and neopterin were assessed at baseline and again at 3 and 6 months after daily ingestion of the respective beverage.

Compared to control, regular consumption of tea over 6 months, did not significantly alter neopterin or tryptophan concentrations, but did result in significantly higher kynurenine (p = 0.016) and Kyn/Trp (p = 0.012). Relative to the control group, in the tea group kynurenine and Kyn/Trp increased during the treatment period by 0.28 μmol/L (95%CI: -0.04, 0.60) and 3.2 μmol/mmol (95%CI: -1.6, 8.0), respectively at 3 months and by 0.48 μmol/L (95%CI: 0.16, 0.80) and 7.5 μmol/mmol (95%CI: 2.5, 12.5), respectively at 6 months.

Increased circulation of kynurenine and Kyn/Trp following regular black tea consumption may indicate enhanced tryptophan breakdown, possibly due to increased activity of immune activation-induced tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). This assumption is supported by the fact that Kyn/Trp correlated with neopterin concentrations throughout the study.

The influence of black tea consumption on biomarkers of immune system activation could relate to its general health benefits and also to its blood pressure lowering property [6] as kynurenine was identified as an endothelium-derived relaxing factor produced during inflammation [7]. Data suggests that net effect strongly depend on the individual immune state, being stimulatory in healthy individuals, while acting more immune dampening in situations with an inflammatory background.

[1] Fuchs D, et al. Curr Med Chem. 2009;16:4644-53.

[2] Pedersen ER, et al. Arterioscler Thromb Vasc Biol 2011;31:698-704.

[3] Zvetkova E, et al. Intern Immunopharmacol 2001;1:2143-50.

[4] Gostner J, et al. Curr Pharm Des 2014;20:840-9.

[5] Gostner JM, et al. Biochim Biophys Acta Clinical 2015;3:31–5.

[6] Hodgson JM &Croft KD. Mol. AspectsMed 2010;31:495–502.

[7] Wang H, et al., Nat Med 2010;16:279–85.

In vitro effects of stabilizer compounds in blood donations

Gostner JM, Becker K, Schennach H, Fuchs D

Biocenter, Medical University, Innsbruck, and Central Institute of Immunology and Blood Transfusion, University Hospital, Innsbruck, Austria

( )

Transfusion-related immunomodulation (TRIM) is controversially discussed as potential risk factor that favors the acquisition of postsurgical infections, the reoccurrence of malignancies and the activation of endogenous viral infections [1]. Otherwise, allogenic blood transfusion was reported to enhance the survival or renal allografts and reduce the recurrence rate of Crohn disease [2]. However, there are only a small number of studies available that discuss such immunomodulatory phenomena and results are often contradictory, and still the blood product constituents responsible for TRIM have not been identified.

In this study we investigated the effect of the individual components of blood storage solutions on indoleamine 2,3-dioxygenase (IDO) activity in unstimulated and mitogen-stimulated human peripheral blood mononuclear cells (PBMC), which has turned out to be a reliable test system to assess potential immunomodulatory properties [3]. The interferon gamma (IFN-γ)-induced enzyme IDO catalyzes the breakdown of the essential amino acid tryptophan to kynurenine, which represents a metabolic checkpoint crucial for pathogen defense but also for immunosuppression [4,5].

Freshly isolated human PBMC obtained from healthy donors were preincubated with different concentrations of blood stabilisation solution (CPD: citrate phosphate dextrose) or red cell preservation solultion components (SAGM: saline, adenine, glucose, mannitol) and were stimulated or not with mitogen phytohemagglutinin (PHA). PHA stimulation activates T cells to produce IFN-γ, which induces IDO activity in monocytes/macrophages. After 48 hours, tryptophan (Trp) and kynurenine (Kyn) levels were determined by HPLC in culture supernatants. IDO activity was indicated by the Kyn/Trp ratio [6]. Cell viability was assessed by analyzing the metabolic activity of cells by measuring the conversion of the substrate resazurin into resorufin.

Glucose treatment of cells did not influence IDO activity in the tested concentration range [11.1- 66.6 mM], but cell viability decreased slightly. Mannitol treatment [0.625-20.0 mM] resulted in a ∼20% decrease of Kyn/Trp ratio and cell viability in PHA-stimulated cells with the highest concentrations. A significant and dose-dependent decrease of Kyn/Trp was measured upon treatment of PBMC with citric acid [6.25-100 mM] in stimulated but not unstimulated cells, e.g., 50.0 mM citric acid addition reduced IDO to 13% of remaining activity, whereas cells viability was 80% of control cells. The higher concentration of 100 mM was already toxic, only ∼15% of both stimulated and unstimulated cells remained viable, pH of the media was lowered to 5.5. Adenine [0.032-100 μM] upregulated IDO activity at lower concentrations slightly but significantly, while the enzyme was suppressed with the highest treatment concentration in stimulated but not in unstimulated cells. Cell viability was also affected, but to a minor extent.

Data indicate the potential of blood storage solutions components to mediate immunomodulatory effects on PBMC in vitro. These findings are in line with our previous report on the effect of erythrocyte concentrates on IDO activity in the same model system, where the SAGM storage solution was identified as a critical contributor [7]. Suppression of IDO activity by several types of antioxidants is discussed as a relevant mechanism to inhibit T helper type 1 reactions, thereby promoting T helper type 2 responses [8].

[1] Cata JP, et al. Br J Anaesth. 2013;110(5):690-701.

[2] Vamvakas EC, Am J Clin Pathol 2006;126(Suppl 1): S71-85.

[3] Becker K, et al. Phytomedicine. 2014;21(2):164-71.

[4]Brandacher G, et al. Curr Opin Organ Transplant. 2008;13(1):10-5.

[5] Munn DH and Mellor AL, Trends Immunol. 34, 137–143 (2013).

[6]Widner B, Adv Exp Med Biol. 1999;467:827-32.

[7]Schennach H, et al. Transfus Med Hemother 2014;41(suppl 1):2–108.

[8] Gostner J, et al. Curr Pharm Des 2014;20:840-9.

Increased neopterin production and tryptophan breakdown in the frail elderly, potential pathophysiologic consequences

Gostner JM, Marcos-Pérez D, Millán-Calenti JC, Sánchez-Flores M, Maseda A, Valdiglesias V, Lorenzo-López L, Strasser B, Pásaro E, Fuchs D, Laffon B

Biocenter, Medical University, Innsbruck, and Institute for Nutritional Sciences and Physiology, UMIT, Hall, Austria; DICOMOSA Group, Department of Psychology, Area of Psychobiology, and Gerontology Research Group, Department of Medicine, Universidade da Coruña, Spain.

( )

Frailty is a geriatric syndrome describing the “weak” physical and mental state of an individual at increased risk for adverse health outcomes. Numerous factors play a role in the development of frailty including immobility (decreased physical activity), loss of balance, depression and cognitive impairment. Phenotypic criteria that indicate compromised energetics include low grip strength, low energy, slowed walking speed, low physical activity, and/or unintentional weight loss [1]. Inflammation could play a major role in the precipitation of characteristic symptoms. An association with increased concentrations of immune system biomarker neopterin has been already described in frail elder individuals [2]. Disturbances in the biosynthesis and reduced availability of specific neurotransmitters like serotonin and adrenalin/noradrenalin are suggested to underlie the high risk of depression and cognitive impairment [3]. With this regard, inflammation-induced breakdown of tryptophan can be of particular relevance. Also, loss of muscle mass and body weight may be a consequence from the developing limitations of this essential amino acid.

In 239 individuals (35 non-frail, 118 pre-frail and 86 frail) aged >63 (mean ± SD: 79.4 ± 8.86) years, measurements of serum neopterin concentrations by ELISA (BRAHMS, Germany) and of tryptophan metabolism by HPLC were performed. The kynurenine to tryptophan ratio (Kyn/Trp) was calculated. Higher neopterin concentrations were associated with higher tryptophan breakdown rates (lower tryptophan and higher Kyn/Trp levels), with frailty status and with older age. The correlations remained significant also after age-adjustment and there was no influence of gender. Higher neopterin and Kyn/Trp as well as lower tryptophan levels were associated with significantly lower grip strength and lower physical activity (all p <0.001).

To summarize, higher age and specific symptoms of frailty are associated with disturbed metabolism of tryptophan. As results of neopterin determinations show, enhanced tryptophan breakdown in frail elderly is associated with immune system activation and implies an influence of cytokine-inducible tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO) on the metabolic changes. Metabolic alterations, especially of tryptophan, could be important for the development of specific symptoms like depression and cognitive deficits in the frail elderly [4].

[1] Fried LP, et al. J Gerontol A Biol Sci Med Sci 2001;56:M146–56.

[2] SX Leng, et al. Age Ageing 2011;40:475–81.

[3] WidnerB, et al. Brain Behav Immun 2002;16:590-5.

[4] Gostner J, et al. Curr Pharm Des 2014;20:840-9.

Impact of resveratrol on tryptophan metabolism in vivo

Gualdoni GA, Mayer KA, Fuchs D, Zlabinger GJ, Gostner JM

Institute of Immunology; Center of Pathophysiology, Infectiology and Immunology, Medical University, Vienna; Division of Biological Chemistry, and Division of Medical Biochemistry, Medical University, Innsbruck, Austria

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Resveratrol is a polyphenol compound found in various nutrients that was shown to have immunomodulatory, anti-cancerogenic, and cardioprotective effects. The regulation of indoleamine-2,3-dioxygenase (IDO), the rate-limiting enzyme in inflammatory tryptophan metabolism, has been proposed to be involved in resveratrol’s biological effects. These observations, however, rely mainly on in vitro findings. We assessed the impact of resveratrol on tryptophan metabolism in a murine sepsis model and in humans after oral resveratrol intake. In the murine sepsis model, balb/c mice were treated with 5 mg/kg bw, 20 mg/kg bw or placebo before inducing a sepsis by LPS administration. We found that resveratrol lowered the kynurenine to tryptophan ratio which is indicative for a decreased IDO activity. This effect was accompanied by an overall decreased inflammatory response as mirrored by a dampened pro-inflammatory cytokine induction. Furthermore, we have analyzed the impact of resveratrol on tryptophan metabolism in humans. Healthy volunteers were orally administrated 5 g resveratrol (n = 8) or placebo (n = 2) in a pilot study. IDO activity was determined by analyzing plasma levels of tryptophan and kynurenine. Determination of the immune activation marker neopterin was included in the analysis. Resveratrol administration resulted in a 1.33- and 1.30-fold increase of the kynurenine to tryptophan ratio at 2.5 h (p <0.01) and 5 h (p <0.01), respectively. Neopterin levels were not affected by resveratrol administration. Concluding, resveratrol differentially affects tryptophan metabolism in situations of induced inflammation and in unstimulated conditions in vivo. Considering the distinct oxidative milieus in these settings, reported findings point towards a mechanism of differential modulation of IDO by resveratrol based on the cellular environment.

Complex connections between marital satisfaction and cellular immune activity in a woman with breast cancer

Hannemann J, Singer M, Primetshofer M, Neises M, Schmid-Ott G, Fuchs D, Ocaña-Peinado FM, Geser W, Schubert C

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

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While studies have shown that troubled marriages can lead to negative immune changes in spouses, a deeper understanding of the complex connections between marital satisfaction and immune system activity is still lacking. This study on a patient with breast cancer sought to investigate the dynamic relation between marital satisfaction and inflammation under as ecologically valid as possible research conditions. The 60-year-old patient (ductal mamma carcinoma diagnosis [pT1c, N0, M0, G2, Her2-/neu receptor] 5 years earlier) collected her entire urine for 31 days in 12-h intervals (from 8 p.m. to 8 a.m. and from 8 a.m. to 8 p.m.; total: 63 measurements) for the determination of urinary neopterin (immune activation marker) and creatinine levels using HPLC. Furthermore, she completed a short questionnaire on marital satisfaction twice each day (at approx. 8 a.m. and 8 p.m.). Cross-correlational analyses revealed that increases in marital satisfaction were followed first by a urinary neopterin concentration increase after 72–84 h (lag 6: 0.263; p <0.05) and then by decreases in urinary neopterin levels after 132–144 h (lag 11: -0.426; p <0.05). We suggest that the long temporal delay between increases in marital satisfaction and decreases in inflammation, as well as the biphasic psychoimmunological response pattern seen in this patient actually point to the dynamic impact of superordinate emotionally positive marital incidents on neopterin production. Such findings once again challenge the usual practice of generalization based on simple data aggregation.

Mycosporine like amino acids, their biological relevance in algae, isolation and pharmacological activities

Hartmann A, Ganzera M

Institute of Pharmacy, Pharmacognosy, Center for Chemistry and Biomedicine, University of Innsbruck, Innsbruck, Austria

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Mycosporine like amino acids, deriving from a broad range of mainly marine algae, cyanobacteria, terrestrial microalgae and even lichens, are small water-soluble molecules with multi-target activities. Recent research revealed MAAs as antioxidants in a variety of different assays such as AAPH and DPPH. The degree of radical scavenging was found to depend on their chemical structure. Hence mycosporine-glycin, an oxo-carbonyl MAA showed much higher antioxidative potential compared to MAAs with cyclohexenimine structure [1-3]. Due to their already known photo-protective properties MAAs might also be potential target molecules for the inhibition of collagenases, whose activity is triggered by UV-light. Therefore, an optimized fluorescent collagenase assay was established and validated to study their biological activity. For this purpose four MAAs were isolated from marine red algae and one lichen by applying different techniques such as ion-exchange chromatography and preparative HPLC. Then they were assayed in-vitro for their inhibition of Chlostridium histolyticum collagenase (Chc). A dose-dependent response was observed for all tested compounds, with IC50 values sometimes lower than the positive control 1,10-phenanthroline (42.86 μg/ml), e.g. that of the MAA porphyra-334 was found to be 37.73 μg/ml.

[1] del la Coba F, et al. J Appl Phycol 2009;21:191-6.

[2] Tao C, et al. Fisheries Sci 2008;74:1166-72.

[3] Rastogi RP & Incharoensakdi A. J Photochem Photob B 2014;130:287-92.

Systematic investigation of the effects of dietary iron supplementation on the course of infection in mice

Haschka D, Demetz E, Tymoszuk P, Asshoff M, Seifert M, Nairz M, Theurl I and Weiss G

Medical University of Innsbruck, Department of Internal Medicine VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, Innsbruck, Austria

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Iron deficiency in children and its sequels growth and mental retardation are an important health issue. Dietary iron supplementation has been considered as a beneficial strategy to overcome iron deficiency, however, in several randomized trials in Africa and Asia iron supplementation was associated with an increased risk of severe infection and infection associated mortality with the mechanisms underlying this devastating observation remaining elusive. We hypothesize that the increased risk of infection in iron supplemented individuals may be related to function of iron as a microbial nutrient and a modulator of anti-microbial immune effector function. Moreover, it is completely obscure up to now which specific group of subjects is at the highest risk for infection upon dietary iron supplementation and whether or not dietary iron intake may be beneficial in another subgroup.

To address these important questions we used different mouse models. To study the impact of baseline iron status (iron adequate versus iron deficiency) on the course of infection we fed mice with a standard rodent diet or an iron deficient diet for four weeks. Subsequently mice were split in two groups, one received dietary/interventional iron supplementation and one group remained on its previous diet. For an acute infection model we used Salmonella typhimurium, an iron dependent bacterium causing severe infection.

We found that after infection with Salmonella for three days, bacterial loads in spleen and liver were highest in previously iron deficient mice which received iron supplementation prior to infection. This phenotype was strictly iron dependent, as infection with a Salmonella mutant strain, which is lacking the three main iron acquiring siderophores resulted in non-significant differences in bacterial loads in organs between the four groups. When analyzing gene expression levels in the spleen of Salmonella infected mice we found a down-regulation of ferroportin expression in spleens of mice with the worst outcome along with a significant increase of IL-10. Correspondingly hepcidin expression levels in the infected livers were proportionally high in worst outcome group.

Our results demonstrate that iron supplementation of iron deficient mice results in an adverse clinical course of infection with the intracellular bacterium S. typhimurium. This is due to low expression of ferroportin which is an important defense strategy of macrophages to restrict these pathogens of iron along with increased expression of the macrophage deactivating cytokine IL-10. Our study will be of helpful in identifying these subjects in areas with a high burden of infection who may or may not benefit from iron supplementation.

Adherence to a Mediterranean dietary pattern and risk of cancer: systematic evidence from observational studies.

Hoffmann, G., Schwingshackl, L.

Department of Nutritional Sciences, University of Vienna, Althanstreet 14 UZA II, A-1090 Vienna, Austria

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The aim of this systematic review was to meta-analyze the effects of adhering to a Mediterranean Diet (MD) on overall cancer risk and risk of different types of cancer. Literature search was performed using the electronic databases MEDLINE, SCOPUS, and EMBASE until 10th January 2015. Only cohort as well as case-control studies were included. Study specific risk ratios (RR) were pooled using a random effect model by the Cochrane software package Review Manager 5.2 (©). 21 cohort studies (1,368,736 subjects), and 12 case-control studies (62,725 subjects) met the objectives and were enclosed for meta-analyses.

The highest adherence to MD category resulted in a significant risk reduction for overall cancer mortality+incidence [cohort; RR: 0.90, 95% CI 0.86 to 0.95, p <0.0001; I2=55%], as well as a significant risk reduction for incidence of either colorectal [cohort/case control; RR: 0.86, 95% CI 0.80 to 0.93, p <0.0001; I2=62%], prostate [cohort; RR: 0.95, 95% CI 0.92 to 0.99, p =0.03; I2=0%], or aerodigestive cancer [case-control studies; RR: 0.34, 95% CI 0.18 to 0.65, p=0.001; I2=76%]. Non-significant changes were observed for breast cancer, gastric cancer, and pancreatic cancer.

According to these results, adherence to a Mediterranean diet is associated with a significant reduction in the risk of overall cancer mortality (by 10%), colorectal cancer (by 14%), prostate cancer (by 4%), and aerodigestive cancer (by 56%), respectively. This is of clinical as well as public health significance, since dietary habits have changed over the past 40 years in favor of a dietary pattern typical for northern European countries. Recommendations to reverse this process, i.e. promoting a Mediterranean dietary pattern typical for southern European regions might prove to be beneficial.

Tryptophan metabolism and its relationship with immune activation, depression, and neurocognitive impairment among HIV-infected individuals

Keegan MR, Chittiprol S Winston A Letendre S, Fuchs D, Boasso A, Iudicello J, Ellis RJ

Imperial College London, London; ViiV Healthcare Ltd., United Kingdom; University of California, San Diego, San Diego, CA, Biocenter, Medical University, Innsbruck, Austria

( )

Neurocognitive impairment (NCI) and major depressive disorder (MDD) are highly prevalent during HIV-1 disease. Two possible mechanisms that could contribute to both NCI and MDD are the accelerated degradation of tryptophan (TRP) via the kynurenine (KYN) pathway, resulting in increased production of neurotoxic catabolites, and the decreased production of serotonin and dopamine, both of which can result from increases in immune activation (IA). We explored whether IA is associated with these two biochemical pathways and their relationship with NCI and MDD in HIV+ individuals.

We performed a cross-sectional analysis of archived CSF and plasma from HIV+ individuals (n=91; receiving antiretroviral therapy [ART], n=65; not receiving ART, n=26) and HIV- controls (n=66), measuring concentrations of IA biomarkers (neopterin [NEO], and tumour necrosis factor [TNF]-α), TRP and KYN, with KYN/TRP ratios as an index of indoleamine 2,3-dioxygenase (IDO) activity, phenylalanine (PHE) and tyrosine (TYR), with PHE/TYR ratios as an index of phenylalanine hydroxylase (PAH) activity, and nitrite, as well as neurocognitive function and mood status. Global Deficit Scores (GDS) were calculated (NCI was defined as a GDS ≥0.5). Non-parametric statistical analyses included Kruskal-Wallis and Mann-Whitney U tests, and multivariate logistic regression.

Groups were comparable for gender and ethnicity. HIV+ participants were older (median age 40 vs. 35; P=0.027) and had higher rates of NCI (39% vs. 14%; P=0.002) and MDD (46% vs. 15%; P<0.001). IA marker concentrations were increased and TRP concentrations decreased in the CSF and plasma of HIV+ participants, including a sub-group of aviraemic HIV+ participants receiving ART (n=44), compared with controls. There were no differences in CSF and plasma KYN between the HIV+ groups and controls. The KYN/TRP ratio in plasma was increased in the total HIV+ group only (P=0.027). The PHE/TYR ratios in CSF, and nitrite concentrations in plasma, were significantly increased in the HIV+ groups. In multivariate analysis, detectable viral loads in CSF and plasma, use of ART and reduced current CD4+ cell counts were predictors of increased concentrations of TNF-α (R2=0.354; P=0.03) and NEO (R2=0.364; P<0.001) in CSF. In a multiple regression model, TNF-α and NEO correlated with KYN/TRP ratios in both CSF (R2=0.277; P=0.008) and plasma (R2=0.674; P<0.001). In a logistic regression model, KYN/TRP ratios in plasma negatively correlated with NCI (χ2[1,n=82]=4.312; B=-1.926; P=0.038), but not with MDD status (χ2[1,n=84]=3.458; B=-1.643; P=0.063) in HIV+ participants. The KYN/TRP ratios in aviraemic participants were negatively correlated with MDD (χ2[1,n=32]=4.874; B=-4.054; P=0.066), but not with NCI (χ2[1,n=32]=3.388; B=-3.078; P=0.066). No associations were observed in viraemic patients or controls. Significant correlations were not observed for PHE/TYR ratios with either NCI or MDD in any of the groups.

In this heterogeneous HIV+ population, we observed increased concentrations of IA biomarkers, decreased TRP, and increases in the KYN/TRP ratio in plasma and the PHE/TYR ratio in CSF. NCI and MDD were associated with a lower KYN/TRP ratio in plasma in both the overall HIV+ group and the aviraemic sub-group. This association was not observed in HIV+ participants with detectable viral loads. The causes of NCI and MDD in HIV+ individuals are likely to be multifactorial and these findings warrant further exploration.

Decreased HDL-cholesterol in still healthy oberweight/obese offsprings indicates parental myocardial infarction and stroke whereas leptin is increased only in male offsprings with positive family anamnesis

Kofler P, Stelzer I, Zelzer S, Reininghaus E, Mangge H

Clinical Institute for Medical and Chemical Laboratory Diagnosis, Austria

( )

Atherosclerosis causes fatal clinical endpoints such as myocardial infarction and stroke. To prevent this, we must improve the understanding of cardiovascular risk factors and early upcoming pathophysiologic mechanisms which can induce clinical useful biomarkers.

Herein we investigated a possible connection between positive family history of cardiovascular events and associated biomarker patterns of offspring.

Study participants are from the prospective, observational study STYJOBS/EDECTA (STYrian Juvenile Obesity Study/Early DEteCTion of Atherosclerosis; ClinicalTrials.gov Identifier NCT00482924) investigating metabolic/cardiovascular parameters in obese young and adults free of chronic health conditions.

We analyzed 15 biomarkers with a possible relation to cardiovascular events in male normal weight, male overweight/obese, female normal weight and female overweight/obese STYJOBS/EDECTA participants. Differences (p<0.05) between participants with a positive family history of myocardial infarction >55years, myocardial infarction <55years, and stroke were compared to a control group with negative family history of both cardiovascular events. Depending on the distribution of the data, all investigations were performed by using the T-Test or the Mann-Whitney U Test.

Male ow/ob participants: In the groups with parental MI, MI<55y and stroke, triglycerides were significantly higher, and HDL-Cholesterol significantly lower compared to the control group without cardiovascular events. Leptin was found significantly higher in MI<55 and in the stroke group.

Male normal weight participants: Significantly higher levels of triglycerides and leptin were seen only in the stroke group. Female ow/ob participants had significantly lower levels of HDL-Cholesterol in the MI and MI<55 group. Female normal weight persons showed no significant differences of any biomarker.

These data underline the importance of decreased HDL cholesterol as an “overall generation” cardiovascular risk factor. All other markers (i.e. CRP, IL-6, LDL, oxLDL, TG, HOMA etc.) failed in this context. Leptin may be of additional interest.

This work was partially supported by funding under the European FP7 program “NanoAthero”-NMP4-LA-2012-3099820.

Osteoprotegerin and RANKL in patients with rheumatoid arthritis

Kurz K, Herold M, Klotz W,Mur E, Weiss G, Fuchs D

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

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Rheumatoid arthritis is an autoimmune disease which is characterized by joint erosions, progressive focal bone loss and chronic inflammation. Pro-inflammatory cytokines like TNF-alpha and interferon-gamma are involved in enhanced bone resorption.

22 patients (20 women, 2 men) with long-standing, moderate to severe rheumatoid arthritis were included in our study and treated with a monoclonal TNF-antibody (Adalimumab 40 mg subcutaneously every other week) in addition to their concomitant but inadequate anti-rheumatic therapies. Patients were assessed for overall disease activity using the DAS28- score and blood samples were collected to determine neopterin, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) concentrations as well as osteoprotegerin (OPG) and soluble Receptor Activator of NF-κB Ligand (sRANKL) concentrations before adalimumab therapy and at week 12. Neopterin as well as OPG and sRANKL were determined by ELISA (neopterin: Brahms, Henningsdorf, Germany, and OPG/sRANKL: Biomedica, Vienna, Austria).

Before adalimumab therapy, neopterin concentrations correlated with OPG (rs = 0.536, p = 0.01) and ESR (rs = 0.552, p < 0.01), and tendentially with CRP (rs = 0.404, p = 0.062), but not with DAS28. sRANKL levels were neither associated with OPG concentrations nor with those of other inflammatory markers.

Upon treatment, disease activity improved significantly (p <0.001), and also CRP and OPG declined significantly (p = 0.023 for CRP, p <0.01 for OPG), while ESR tended to be lower (p = 0.086). However, there were no significant changes of neopterin, sRANKL and the sRANKL/OPG ratio. After 12 weeks of adalimumab therapy, the correlation between OPG and neopterin was not detectable anymore.

We conclude that therapy with adalimumab was very effective to modify disease activity (as reflected by the decrease in the number of tender and swollen joints) and also slow-down inflammatory and bone remodelling cascades.

Pre-, pro- and psychobiotics in old age

Leblhuber F, Schuetz B, Fuchs D

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

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Gastrointestinal health is of increasing interest in preventive medicine. It includes normal intestinal microbiota and effective immune status. Gastrointestinal barrier function and gut microbiom seem to be the key to good health.

Prebiotics are nondigestible food ingredients that beneficially effect the host by selectively stimulating the growth and activity of bacteria in the colon and thus improve host health that is especially important in the elderly, as there is an age-related decrease in the diversity of gut microbiota.

Probiotics are living organisms, which when administered in adequate amounts confer health benefits on the host: probiotics suppress pathogens, stimulate epithelial cell proliferation, differentiate and fortify the intestinal barrier and mediate immmune modulation. However, there is still confusion in the minds of the authorities over whether a probiotic is a drug, a food, or a dietary supplement.

Gut microbes can produce hormones and neurotransmitters like short chain fatty acids, g-amino-butyric acid (GABA), serotonin, tryptophan and other biogenic amins like adrenalin and noradrenalin that are identical to those produced by the host. Therefore such probiotics are also named psychobiotics, which act on the brain-gut axis. The effects are mediated via the vagus nerve, spinal cord and the hypothalamic-pituitary-adrenal axis.

Dysbiosis (perturbation in microbiom composition) results in dysbalance of neurotransmitters and consecutively in depression, anxiety, altered pain perception and other symptoms like cognitive decline and dementia [1].

Administration of prebiotics and probiotics like Omnibiotic (Allergosan, Graz, Austria) may alter the gut microbiom and improve the disturbed resorption of neurotransmitters important for the neuropsychiatric status in the elderly.

[1] Leblhuber F, et al. J Neural Transm 2015, Feb 14, DOI 10.1007/s00702-015-1381-9.

Routine serum and urine neopterin measurement in a clinical central laboratory

Loacker L, Weigel G, Griesmacher A

Central Institute for Medical and Chemical Laboratory Diagnostic, University Hospital, Innsbruck, Anichstraße 35, Austria

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Neopterin is a biologically stable and helpful marker reflecting the cell-mediated immunity and is therefore able to enfold the state of activation of the entire immunological system. Moreover it provides an opportunity to monitor and estimate the prognosis of multiple diseases such as infections, autoimmune diseases, malignancies, cardiac and renal failure or allograft rejection. In routine laboratories neopterin can be determined by ELISA, RIA or HPLC.

In a retrospective examination we investigated the quantity and numeric development of all neopterin analyses in the period of 2008 to 2014 at the Innsbruck University Hospital, the frequency of different underlying diagnoses and the distribution of requesting medical departments. neopterin was determined in human serum and urine samples using an ELISA or RIA assay in a routine laboratory setting.

There is a distinct increase of 34% of urine and 198% of serum neopterin measurements comparing the analysis frequency in 2008 and 2014. Main areas of clinical interests for determination actually are infectious diseases, HIV monitoring and autoimmune diseases. It is therefore most frequently requested by the infectiology, gastroenterology and dermatology departments.

Neopterin measurement is widely established at the Innsbruck University Hospital. Beside its known value in the assessment of severity, course, activity and treatment control of immunological diseases neopterin is also getting relevant in recent clinical fields of application such as cardiovascular or metabolic diseases.

Improved diagnosis and stabilization of vulnerable atherosclerotic plaques – a main challenge to fight myocardial infarction and stroke

Mangge H, Almer G, Opriessnig P, Reininghaus E, Prassl R

Clinical Institute for Medical and Chemical Laboratory Diagnosis, Medical University, Graz, Austria

( )

Vulnerable atherosclerotic (AS) plaques cause fatal clinical endpoints such as myocardial infarction and stroke. To prevent this, we must improve the early diagnosis and treatment of these fatal vascular lesions. Because vulnerable AS plaques are frequently non-stenotic, they are preclinical undetectable by conventional imaging. Blood lipids, C-reactive protein, and interleukin-6 are insufficient to indicate the process of critical perpetuation prior to endpoints. More specific biomarkers (e.g., troponin, copeptin, natriuretic peptides, growth differentiation factor-15, soluble ST2) indicate acute coronary syndrome or cardiac insufficiency, but not critically destabilized AS lesions. Thus, valuable time (months to years) that could be used to treat the patient is wasted. An improved management of this dilemma may involve better detection of variations in degrees of immune inflammation in plaques by using new biomarkers in blood and/or within the lesion by nanotechnological aided molecular imaging.

Macrophage and T-cell polarization, innate- and adaptive immune responses (e.g., The Toll-like receptors 2, 4, 7), are involved in the process of plaque “vulnerabilisation”. New biomarkers involved in these mechanisms include adiponectin, interleukins, pentraxin 3, calprotectins S100A8/A9, myeloperoxidase, and chemokines. These proteins may be candidates for molecular imaging by using NMRI/nuclear imaging tools.

We found out recently that globular adiponectin or interleukin-10 targeted pegylated “stealth” liposomes are promising nanoconstructs for molecular imaging of AS lesions in animal models (ApoE mice, rabbits) by means of NMRI. Under the scope of the EU project NanoAthero this approach is examined for a development to the human pipeline.

Nevertheless, the main challenge remains: which asymptomatic individual should be screened? At which time? Furthermore, it is essential to act specific, effective, without side effects because the “patient” may yet feel healthy at the time of the successful prediagnosis.

This work was partially supported by funding under the European FP7 program “NanoAthero”-NMP4-LA-2012-3099820.

IDO1 controls anti FVIII antibody response in hemophilia A

Matino D, Gargaro M, Santagostino E, Di Minno D, Landolfi R, Morfini M, Rocino A, Mancuso ME, Di Minno MND, Coppola A, Volpi C, Vacca C, Iannitti R, azzucconi MG, Santoro C, Tosti A, Tagariello G, Belvini D, Radossi P, Castaman G, Fuchs D, Boon E, Grohmann U, Puccetti P, Iorio A, Fallarino F

Department of Experimental Medicine, University of Perugia, Perugia, Italy; Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico Milan, Italy; Regional Reference Centre for Coagulation Disorders, Department of Clinical and Experimental, Medicine, Federico II University, Naples, Italy; Institute of Internal Medicine and Geriatrics, Haemostasis Research Center, Catholic University, Rome, Italy;Centro Emofilia, Azienda Ospedaliera Careggi, Florence, Italy; Haemophilia and Thrombosis Centre, San Giovanni Bosco Hospital, Naples, Italy: Department of Cellular Biotechnology and Haematology, La Sapienza University, Rome, Italy. Department of Medicine University of Perugia, Perugia, Italy; Transfusion Service, Hemophilia and Regional Blood Disease Centre, Castelfranco Veneto, Italy; Dept. of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy; Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; Bioceros, Utrecht, The Netherlands; Departments of Clinical Epidemiology and Biostatistics and Medicine, McMaster University, Canada

Inhibitory antibodies to FVIII are a major obstacle to hemophilia A treatment. Congenital absence of FVIII prevents onset of central tolerance to FVIII, thus foisting effective control of FVIII-reactive lymphocytes on peripheral tolerance mechanisms. Indoleamine 2,3 dioxygenase-1 (IDO1) is a key regulatory enzyme of peripheral tolerance in adult life. We investigated the association between IDO1 competence and inhibitor status in hemophilia A patients, enrolled in six Italian centers, with F8 null mutations and either an inhibitor-negative (N = 50) or positive (N = 50) status. By analyzing IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression, we found that defective IDO1 induction by Toll-like receptor (TLR)9 activation was associated with an inhibitor-positive status. In an experimental model of the disease using hemophilic mice with or without functional IDO1, we found that tryptophan metabolites resulting from IDO1 activity prevented the generation of anti-FVIII antibodies and that a TLR9 agonist caused suppression of FVIII-specific B cells by a mechanism involving IDO1-dependent induction of regulatory T cells. Thus IDO1 competence may represent a novel patient risk stratification tool in hemophilia A, thus implementing novel strategies for preventing or eradicating inhibitors to therapeutically administered FVIII protein.

Kynurenine metabolites in acute alcohol withdrawal

Mechtcheriakov S, v. Gleissenthall G, Arnhard K, Oberacher H

Department of Psychiatry and Psychotherapy, and Institute of Legal Medicine and Core Facility Metabolomics, Medical University, Innsbruck, Austria

( )

Recent research has suggested that alcohol-associated immune activation may contribute to the pathophysiology of alcohol withdrawal and dependence. We studied the relationships between an immune activation marker neopterin and parameters of tryptophan and kynurenine metabolism during the first 10 days of alcohol withdrawal in alcohol dependent patients.

Our results show a significant correlation between serum neopterin values and kynurenine/tryptophan ratio as indicator of IDO-activation (r = 0.695, p <0.001). Serum levels of quinolinic acid (QA) were also strongly related to neopterin concentrations as measured by linear mixed model (p<0.001) while the kynurenic acid (KYNA) concentrations showed no relationship with neopterin (p=0.929).

These findings demonstrate (1) an immune-triggered activation of IDO in acute alcohol withdrawal causing the increase of kynurenine production and suggest (2) that immune activation also stimulates the production of quinurenic acid that is known to exert glutamatergic (potentially excitotoxic) effects. These data emphasize the specific role of the immune activation in regulation of tryptophan and kynurenine metabolism in alcohol disease.

Prognostic significance of urinary neopterin compared with neutrophil-to-lymphocyte, lymphocyte-to-monocyte and platelet-to-lymphocyte ratios in patients with breast cancer

Melichar B, Zezulová M, Bartoušková M, Kalábová H, Študentová H, Vitásková D, Kujovská Krčmová L, Solichová D

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

( )

Predictive and prognostic biomarkers play and increasingly important role in the management of cancer patients. The significance of inflammatory reaction that reflects the host response to neoplastic growth is being increasingly recognized. A number of biomarkers of the inflammatory response have been introduced into the management of cancer patients over the last decades, including, for example, C-reactive protein (CRP) and neopterin. In the last few years considerable research has been focused on simple biomarkers obtained by calculating the ratios of lymphocytes to other cellular components of peripheral blood. The neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and platelet-to-lymphocyte ratio (PLR) have been demonstrated to represent independent prognostic biomarkers across a spectrum of solid tumors. We performed a retrospective analysis of previously published cohorts of patients with history of breast cancer. NLR, LMR and PLR were calculated from peripheral blood cell counts obtained as part of routine care. Survival data were updated for 2 cohorts of patients (n=72 and n=474). Urinary neopterin was determined by high-performance liquid chromatography. LMR calculated based on automated and manual counts was significantly different. LMR and PLR were higher in patients compared to controls. A significant correlation was observed between peripheral blood cell counts (PBC)-derived ratios, but no correlation was noted between urinary neopterin and PBC-derived ratios. In a cohort of 72 patients NLR≥3, but not urinary neopterin was significant predictor of survival. In contrast, in an unselected cohort of 474 patients urinary neopterin and NLR≥3.5 were significant predictors of survival. In multivariable analysis, age, hemoglobin, urinary neopterin and NLR were independent predictors of survival. In conclusion, PBC-derived ratios did not correlate with urinary neopterin. In different cohorts, both NLR and urinary neopterin were significant predictors of survival.

Investigating the NAD metabolome in Ewing Sarcoma

Mutz Cornelia N, Schwentner Raphaela, Kauer Maximilian O, Ban Jozef, Aryee Dave NT, Erhardt Sophie, Fuchs Dietmar, Heitger Andreas,* Kovar Heinrich

Children’s Cancer Research Institute and Department of Pediatrics, Medical University, Vienna, Austria; Department of Physiology and Pharmacology, Karolinksa Institutet, Sweden; Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; *deceased

Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in generation of the chimeric transcription factor EWS-FLI1.

Recently, ES has been reported to be exquisitely sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1). This enzyme uses NAD as substrate and was demonstrated to regulate EWS-FLI1 in a feed-back mechanism. NAD is a key metabolite essential for sustaining cellular energy metabolism. It plays a central role in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. The other major mammalian NAD consumer is the deacetylase SIRT1 which we observed to be specifically highly expressed in ES metastases. PARP1 and SIRT1 are crucial for coupling cellular metabolism to transcriptional gene regulation as well as to stress response. Usually, NAD is regenerated from nicotinamide in the NAMPT-dependent salvage pathway or from the reduction of pyruvate via LDHA (Warburg effect), but can also be synthesized de novo from tryptophan. Interestingly, the knockdown of EWS-FLI1 in ES cells comes along with alterations in the expression of multiple enzymes involved in NAD biosynthesis and regeneration, including TDO2, KMO, NMNAT1, NAPRT, NAMPT, and LDHA. We are interrogating the role of EWS-FLI1 mediated modulation of these enzymes and of specific small molecule inhibitors on cellular tryptophan consumption, kynurenine production and intracellular NAD levels of Ewing sarcoma cells. Additionally, we are investigating the functional consequences thereof on constitutive and stress-induced protein poly(ADP)-ribosylation and acetylation.

Preliminary results suggest that NAMPT inhibition diminishes PARP1 activity due to low NAD, implying a pivotal role for the regenerative salvage pathway in ES cells. TDO2-dependent tryptophan consumption increases in the absence of the oncogene. These studies aim at a better understanding of factors influencing Ewing sarcoma sensitivity to therapies targeting PARP1 and the NAD metabolome.

Supported by the Austrian Science fund, grant I1225-B19.

Comparison of LC/MS and LC coupled with UV and fluorescence detection for the analysis of tryptophan metabolism

Oberacher H, Arnhard K, Pitterl F, Sperner-Unterweger B, Fuchs D

Institute of Legal Medicine and Core Facility Metabolomics, Department of Psychiatry and Psychotherapy, and Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Austria

( )

The kynurine pathway is the most important pathway in tryptophan (TRP) catabolism which accounts for more than 90% of TRP breakdown in mammals. Important TRP metabolites include kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN), and they are known to be active in the nervous and/or immune systems. For studying the physiological roles of TRP and its metabolites, the availability of reliable, sensitive and comprehensive quantitative methods is of utmost importance. Such analytical techniques are liquid chromatography with UV and fluorescence detection (LC) as well as liquid chromatography coupled to high resolution mass spectrometry (LC/MS).

The LC method used for comparison in this study [1] enables the accurate and reproducible quantification of TRP and KYN: 100 μl of plasma are prepared for reversed-phase LC analysis by protein precipitation with trichloroacetic acid, 3-nitro-L-tyrosine is added as internal standard. Aqueous solutions of TRP spiked with 70 g/l albumin are used as surrogate matrix for preparing calibration standards. Ten μl of produced supernatants is injected on a reversed-phase column. Run time is 7 min. Quantification is based on peak height comparison to an external standard. The LC method was validated. Reproducibility was found to be <5%. Recoveries were 99% for TRP and 102% for KYN.

To enable the quantification of TRP and its three metabolites, a LC/MS method was developed. 50 μL of plasma are prepared for LC-MS analysis by protein precipitation with acetonitrile. Deuterated analogues of TRP, KYNA, and QUIN serve as internal standards. TRP metabolite-free plasma obtained from treatment with activated charcoal is used as surrogate matrix for preparing six calibration standards and two quality control samples. Analytes are separated on a HILIC column using a gradient of 50-5% acetonitrile in aqueous ammonium formate (5 mM, pH 9.5), and detected on a quadrupole – quadrupole – time-of-flight instrument (TripleTOF5600+, ABSciex, Foster City, CA, USA) using electrospray ionization (ESI) in negative ion mode. The acquisition strategy involves the collection of full scan MS/MS spectra and post-acquisition extraction of analyte-specific fragment ion mass traces. Run time is 25 min. The developed method was validated. The parameters tested for each compound included calibration model, limit of quantification, accuracy, reproducibility, sample stability, recovery, and matrix effects. The assay was well within tolerances prescribed by regulatory guidance for validation of quantitative LC/MS assays.

89 samples were analysed with both methods available. TRP and KYN levels showed good correlation. The Spearman’s rank correlation coefficients were found to be 0.87 and 0.67.

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

Plasma levels of tyrosine, phenylalanine, tryptophan kynurenine and neopterin in chronic lymphocytic leukemia (CLL) patients

Parrak V, Secnik P, Mistrik M, Melichar B, Galba J, Michalicova A, Kovac A, Katina S, Fuchs D

University Hospital, and Slovak Academy of Science, Bratislava and SK-Lab, Lucenec, Slovak Republic; University Hospital Olomouc and Masaryk University, Brno, Czech Republic; Biocenter, Medical University, Innsbruck, Austria

( )

Chronic lymphatic leukemia (CLL) is the most common leukemia in adults in the Western world with highly variable clinical course, with unclear prognosis and still uncurable. A lot of studies investigated predictive markers which can be helpful for predicting the individual risk in different stage of the disease, or to find a diagnostic marker or marker which can follow up the course of therapy. The immune system plays an important role in CLL and immunological markers shows changes in their levels in disease follow up.

There are some early experiences with neopterin determinations in urine (1). Hausen et al. and our own experiences which show significant elevation of neopterin in plasma in patients with CLL in more than 200 patients (about 600 plasma samples) within 3 years of observation.

We have collected EDTA plasma samples from blood sampling upon routine examinations (blood count) aliquotted samples are stored at -80o C. Neopterin was analysed by Enzyme-Linked Immunosorbent Assay

(ELISA) BRAHMS, Henningsdorf, Germany with a reference value of 5.3 ± 2.7 nmol/L; kynurenine, tryptophan, phenylalanine, and tyrosine concentrations were determined by UPLC/FLD/MS Waters. These parameters were measured in 75 EDTA plasma samples (34 females, 31 males) with same diagnosis, similar age, collected in 1 month from blood count, aliquotted and stored at -80o C.

Our first results of statistical analyses allow the following preliminary conclusions however, from a small number of samples only: On average, CLL patients show increased plasma neopterin concentrations and compared to healthy controls (2) also the kynurenine to tryptophan ratios (Kyn/Trp) and the phenylalanine to tyrosine ratios (Phe/Tyr) were increased. Moreover, there existed significant correlations between neopterin and Kyn/Trp as well as Phe/Tyr.

We also found some gender differences in amino acids related. New questions about the influence of age and of therapy will be addressed during further statistical analyses.

[1] Hausen A, et al. Clin Biochem 1982;15:34-7.

[2] Geisler S, et al. Pteridines 2015;26:31–6.

Laboratory diagnostic markers: is there a potential in diagnosis of Alzheimer’s disease?

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

Axon-Neuroscience SE and Slovak Academy of Science, Neuroimmunological Institute, Bratislava, and SK-Lab, Lucenec, Slovak Republic; and Masaryk University, Brno, Czech Republic

( )

Alzheimer’s disease (AD) is a chronic, irreversible neurodegenerative disease that causes progressive impairment of memory and cognitive function. AD is a leading cause of dementia worldwide. Epidemiological data about dementia (1) reveals that currently 20 to 30 million individuals suffer from dementia today, with 4.6 million new cases of dementia every year. Neurofibrillary degeneration represents one of the key hallmarks of AD. In spite of intensive research, there is no disease modifying drug for AD treatment available. The therapeutic intervention is considered to be most effective when administered early in the disease’s progression, before neurodegeneration is too severe and widespread through the brain.

The great effort is spent in a search for novel diagnostic tools or laboratory markers that accurately identify AD in the very early phase of the disease progression. Such marker must be sensitive to earliest changes in AD and should differentiate among preclinical AD, normal aging and other brain disorders that cause memory loss. We have developed the first-in-man and first-in-class tau vaccine for AD denominated as AADvac1. AADvac1 immunotherapy is currently in Phase 1 clinical trial in four hospitals in Austria. According to our first results, AADvac1 has a favorable safety profile and acceptable antibody response in patients. Samples of human biological fluids were collected at every hospital visit and banked. In the future, this collection should represent the unique opportunity to search for novel AD diagnostic tool or laboratory markers.

[1] Ferri CP, et al. Lancet 2005;366:2112-7.

Neopterin as a monitoring marker in patients with high-risk, non-muscle invasive bladder cancer and intravesical bacillus Calmette-Guèrin (BCG) therapy: a pilot study

Pichler R, Fuchs D, Fritz J, Brunner A, Horninger W, Culig Z

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

( )

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 [1-2].

Due to the fact that both biochemical pathways (neopterin production) 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 and urinary neopterin 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]. Since September 2014, 8 (2 women, 6 men) patients with a mean (range) age of 76 (65-89) years who were treated once a week with intravesically instilled BCG according to the empirical 6-weekly schedule were reviewed prospectively. Histology confirmed primary carcinoma in situ (CIS) in 1 (12.5%), urothelial carcinoma pTa high grade in 4 (50%), pT1 high grade in 2 (25%) and pTa+pT1 high grade in another 1 (12.5%) patient. 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 months after BCG induction therapy. Cystoscopy 3 months after completed BCG induction therapy confirmed cancer recurrence (pTa low grade) in 2 (25%) of 8 patients. Serum neopterin concentrations were lowest in patients with cancer recurrence (n=2) before (median, range: 6.9, 5.4-8.4 nmol/L), during (median after BCG1: 9.6 nmol/L, after BCG2: 7.95 nmol/L, after BCG3: 8.7 nmol/L, after BCG4: 8.85 nmol/L, after BCG5: 8.15 nmol/L; after BCG6: 10.55 nmol/L) and 3 months after BCG therapy (median, range: 7.25, 7.1-7.4 nmol/L) compared to those patients without recurrence (n=6): before (median, range: 12.9, 6.1-14.4 nmol/L), during (median after BCG1: 12.0 nmol/L, after BCG2: 11.3 nmol/L, after BCG3: 12.2 nmol/L, after BCG4: 13.8 nmol/l, after BCG5: 14.5 nmol/L, after BCG6: 15.05 nmol/L) and 3 months after BCG induction (median, range: 16.6, 5.8-21.8 nmol/L). In addition, urinary neopterin levels were higher in recurrence-free patients (median, range: 201, 124-305 μmol/mol creatinine) compared to those patients with cancer recurrence (median, range: 95.5, 51-140 μmol/mol creatinine) before, during and 3 months after (median, range: 344, 127-384 versus 149, 144-155 μmol/mol creatinine) BCG therapy.

From this preliminary data obtained serum and urinary neopterin could be a potentially promising and easily reproducible monitoring marker in patients with high-risk NMIBC and intravesical BCG therapy. However, these incidental findings must be further studied in a larger prospective trial prior to drawing any 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.

Determination of the neopterin concentration- is it a useful parameter in the pediatric department?

Plata- Nazar K, Luczak G, Kaminska B

Department of Pediatrics, Pediatric Gastroenterology, Hepatology and Nutrition, Medical

University of Gdansk, Gdansk, Poland

( )

Evaluation of the usefulness of clinical parameters of inflammation in clinical practice is still a topical issue. These parameters are routinely performed in the pediatric departments, in almost every patient. Commonly identified are: blood count and differential white blood cell count, erythrocyte sedimentation rate (ESR), C- reactive protein (CRP) and procalcitonin (PCT) level in serum. All of these parameters are non-specific. Erythrocyte sedimentation rate study is imprecise, lazily rising and slowly returns to normal. Highly increased level of CRP- acute phase protein- is observed mainly in the course of bacterial infections. PCT is an early marker of systemic bacterial infections. Therefore researchers are looking for more specific parameters of viral infections which could be applied for routine clinical practice. One of these parameters seems to be neopterin (NPT) level. Elevated levels of neopterin are observed in diseases in which cellular immune response is stimulated. Monitoring changes of the immune system in the course of illness, by means of a rapid and sensitive method, helps to explain the disease etiology and gives the ability to implement appropriate treatment.

The study group consisted of 363 children with different inflammatory diseases. The control group consisted of 105 healthy children. The serum neopterin concentration was analyzed by immune-enzymatic method (ELISA) using a commercial test set (Brahms, Hennigsdorf, Germany). Based on the results obtained in the control group, the reference level of NPT in serum was estimated as 11nmol/L, regardless of age and gender. In our studies determination of neopterin concentration was considered as a useful parameter in differential diagnosis of infections etiology. High levels of NPT were observed in viral and intracellular bacterial infections while normal levels of NPT are characteristic for other typical bacterial infections. Determination of NPT was considered as a useful parameter in monitoring progress, course and treatment of autoimmune diseases (inflammatory bowel disease, rheumatoid disease).

There was a significant positive correlation between serum levels of NPT and CRP only in the group of children with inflammatory bowel disease. In other groups of children correlation between NPT and CRP levels was not observed. There was also no significant correlation between NPT and PCT.

Determination of serum NPT level, as parameter independent of other markers of inflammation, is valuable for the diagnosis of inflammatory diseases in children.

Toxoplasma gondii seropositivity and high kynurenine levels are associated with suicide attempts in patients with schizophrenia: Potential interaction with stress induced inflammation

Postolache TT, Okusaga O, Duncan E, Langenberg P, Brundin L, Groer MW, Giegling I, Hartmann AM, Konte B, Friedl M, Rujescu D, Fuchs D

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) 5, Mental Illness Research Education and Clinical Center (MIRECC), Baltimore, MD

( )

Both Toxoplasma gondii (T. gondii) chronic “latent” infection, and molecules of the kynurenine pathways (kynurenine and quinolinic acid), have been previously associated with suicidal self-directed violence. As infection with T. gondii is known to elevate kynurenine levels, and because high kynurenine levels are in turn immunosuppressive, thus potentially contributing to reduced immune pressure and induce reactivation of T. gondii, we hypothesized that T. gondii association with suicide attempt history will be stronger in those with high kynurenine levels. 950 patients with schizophrenia were evaluated clinically, anti-T.gondii IgG antibodies were measured with using solid-enzyme immunoassay as well as kynurenine levels using with high performance liquid chromatography. In T. gondii seropositive patients only, KYN levels in the upper 25th percentile were associated with nonfatal suicidal self-directed violence. The results argue against a linear mediation of T. gondii effect by kynurenine and its metabolites. We propose that the immunosuppressive effect of high levels of kynurenine, resulting from a dysregulated kynurenine pathway, may contribute to T. gondii reactivation, and suicidal behavior. If confirmed in future longitudinal studies, this result may have both theoretical and practical treatment implications. Additionally, interactions with stress induced activation

Supported by an American Foundation for Suicide Prevention Distinguished Investigator Award (PI Postolache) with additional support from the Rocky Mountain MIRECC, Denver, Co. Drs Postolache and Rujescu equally contributed to this project.

In psychiatrically healthy individuals, overweight women but not men have lower tryptophan levels

Postolache TT, Raheja UK, Giegling I, Mohyuddin I, Rovner SF, Weghuber D, Mangge H, Rujescu D, Fuchs D

Mood and Anxiety Program, Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, USA; Psychiatry Residency Training Program, Saint Elizabeths Hospital, Washington, DC, USA; Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; Department of Psychiatry, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany; Frontier Medical Group, El Paso, TX, USA; Department of Pediatrics, Paracelsus Medical School, Salzburg, Austria; Research Unit on Lifestyle and Inflammation-associated Risk Biomarkers, Clinical Institute for Medical and Chemical Laboratory Diagnosis, Medical University of Graz, Graz, Austria

Teodor T. Postolache, and Dan Rujescu equally contributed to this project

Obesity has been shown to be associated with elevated kynurenine (KYN) and kynurenine-tryptophan (KYN/TRP) ratio, an index estimating tryptophan breakdown rate. Previous research showed that obese women are more likely to be depressed than obese men. To exclude effects of depression on tryptophan breakdown, we repeated the above investigations in psychiatrically healthy individuals. In 1000 participants, plasma KYN, TRP, and their ratio were compared between overweight/obese and normal-weight individuals using ANCOVA with adjustment for age and gender and bivariate post-hoc tests.

There was no significant relationship between KYN, TRP, and KYN/TRP ratio and overweight/obese status. A significant gender by weight category interaction was identified for TRP only, with overweight/obese women having lower TRP than overweight/obese men (p = 0.02). The results suggest that the previously described association between obesity and KYN may have been mediated by depression or other psychopathology. Our results also affirm that lower TRP levels in obese women are not secondary to depression (e.g., via indoleamine 2,3-dioxygenase activation), that multiple vulnerabilities are required for obesity related depression in women, and that manipulations of TRP levels could be used to advance theoretical knowledge and therapeutic control of depression in obese women.

The expression of cathelicidin gene is reduced in the prefrontal cortex of depressed suicides

Postolache TT, Zhang H, Lee E, Streeten E, Brenner LA, Dwivedi Y

University of Maryland, Baltimore, MD, Rocky Mountain MIRECC, Denver CO, University of Alabama School of Medicine, Birmingham, AL

Severe hypovitaminosis D leads to immune dysregulation, increased vulnerability to infections, and is associated with suicidal self-directed violence (SSDV). Vitamin D stimulates the production of cathelicidin, an antimicrobial peptide identified in humans in the skin. As brain cathelicidin-homologues were previously located in rodents, we endeavored to uncover brain cathelicidin gene (CAMP) expression in humans. We further hypothesized that among depressed suicides, brain CAMP expression is decreased and of vitamin D receptor gene (VDR) is increased. Expression levels of CYP24A1 and CYP27B1, metabolic genes critical for the local production of activated Vitamin D were also measured.

Expression levels of CAMP, Vitamin D receptor (VDR) genes, CYP24A1and CYP27B1 in the dorsolateral prefrontal cortex (dlPFC) and anterior cingulate cortex (ACC) were compared between depressed suicides (n=15), and matched (age, gender, and postmortem-interval) non-psychiatric controls (n=15). Gene expression was analyzed with qRT-PCR with TaqMan® primers and probes, with GAPDH and β-actin genes as endogenous controls. Statistical analyses included ANOVAs with post-hoc t-tests with Bonferroni corrections.

CAMP expression was significantly down-regulated and VDR expression significantly up-regulated (p <0.05 corrected) in dlPFC and ACC In depressed suicides, with no significant differences in CYP24A1 or CYP27B1 expression. Future studies should include other brain regions, larger N, psychiatric controls, protein measures, and tissue localization with in-situ hybridization. Elevated VDR and lower CAMP gene expression in depressed suicides is consistent with associations of depression and SSDV with hypovitaminosis D, inflammation and infection.

The study was partly funded by R01MH082802 (Y. Dwivedi), by the American Foundation for Suicide Prevention (T.T. Postolache) and the Rocky Mountain MIRECC, Denver, CO (T.T.Postolache, L.A. Brenner). Drs. Postolache and Dwivedi equally contributed to this project.

The role of telomeres in cellular aging: effect of vitamins supplementation on LINE-methylation and telomere length in elderly

Pusceddu I, Herrmann W, Kirsch S, Obeid R, Hübner U, Geisel J, Herrmann M

Department of Clinical Chemistry, Saarland University Hospital, Homburg, Germany; Department of Clinical Pathology, District Hospital Bolzano, Italy

( )

Telomeres are essential for the maintenance of genomic integrity. Telomere length declines with age and telomere shortening/dysfunction has been proposed as biomarker for age-related diseases. B and D vitamins are essential cofactors for numerous cellular processes including the synthesis of purines and nucleotides, DNA methylation, cell differentiation, proliferation and apoptosis. B and D vitamin deficiencies are risk factors for the development of age-related diseases. The aim of this study was to evaluate the effects of B and D vitamin supplementation on telomere biology in healthy elderly people.

In a double-blind study 60 subjects (>54 years) were randomly assigned to receive a daily combination of vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg) and calcium carbonate (456 mg) (Group A) or vitamin D3 and calcium carbonate alone (Group B). Blood concentrations of 25-hydroxy-vitamin D, vitamin B12, vitamin B6, folate and several metabolites were measured. Furthermore, LINE-methylation and telomere length in peripheral blood leucocytes were analyzed at baseline and after 1 year of supplementation.

Baseline gender- and age-adjusted telomere length correlated with 5-methyl-tetrahydrofolate (r=0.35) and total folate (r=0.33). At the end of the study gender- and age-adjusted telomere length showed the following correlations: Group A: methylmalonic acid (r=-0.46) and choline (r=0.39); Group B: 5,10-methenyl-tetrahydrofolate (r=-0.57), dimethyl-glycine (r=-0.39), and LINE-1 methylation (r=-0.43). The significant increase of choline and decrease of methylmalonic acid after one-year treatment with B and D vitamins was significantly related to improved telomere length.

The present results suggest a functional relationship between vitamin B status and telomere length in elderly subjects. One-year treatment with B and D vitamins significantly changed the pattern of correlations observed at baseline. The improved vitamin B12 status after vitamin supplementation showed a significant relation to telomere length. Moreover, a sub-optimal vitamin B status may result in telomere dysfunction through altered DNA methylation.

Quantum chemical studies on pterin radicals

Reibnegger G

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

( )

Pteridines are an important class of heterocyclic molecules which play important roles in many biological systems. In particular, 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 the electronic nature of such systems appears of considerable interest.

Using reliable quantum chemical computations, I present a report on the electronic structure of neutral radicals formally derived from pterin by abstraction of one of the five hydrogen atoms.

All calculations are done in gas phase. Starting with the molecular geometry of neutral pterin in its most stable tautomeric form [3,4] optimized using density functional theory (B3LYP/6-31G(d)), the five potential radicals are built by deleting one of the hydrogen atoms. For each pterin radical, a new geometry optimization is performed, followed by a vibrational analysis in order to ensure that a true minimum structure is found. Using this optimized structure, a more accurate single-point computation at the B3LYP/6-311+G(2d,p) level is done yielding the molecular wave function. The electron density, the spin density and the electrostatic potential (ESP) functions are obtained in cubes surrounding the molecules and employing a 192x192x192 grid. These calculations are 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) are 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 is done with AVS Express 5.3 software (Advanced Visual Systems Inc., Waltham, MA, USA).

The results demonstrate that the five possible radicals derived from neutral pterin fall into two markedly different classes: abstraction of one of the three hydrogen atoms bonded to a nitrogen atom yields radicals exhibiting a strong delocalization of the unpaired electron over both the pyrimidine and the pyrazine part of the pterin system, while the radicals lacking one of the two carbon bonded hydrogen atoms show strong localization of the spin density at the affected carbon atoms without any delocalization via the p-electron system. Hence, the latter two radicals are somewhat less stable than the resonance-stabilized variants. Further differences between the radicals in terms of planar versus non-planar geometries, strengths of the chemical bonds, ESP and ELF are discussed.

The presented calculations provide a basis for further investigations, including also the biologically important dihydro and tetrahydro pteridine derivatives as well as anionic and cationic radicals.

[1] Öttl K, et al. Helvetica Chimica Acta 2000;83:954-65.

[2] Lorente C, et al. Pteridines 2011;22:111-9.

[3] Soniat M & Martin CB. Pteridines 2008;19:120-4.

[4] Reibnegger G. Pteridines 2014;25:41-8.

Alkylglycerol monooxygenase in 3T3-L1 adipocyte differentiation

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

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

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Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin-dependent transmembrane protein and is the only enzyme capable of metabolizing alkylglycerols. However very little is known about its physiological role. Our laboratory was able to identify the sequence coding for alkylglycerol monooxygenase in 2010 [1]. Its expression and activity can be detected in a variety of tissues and cell lines including adipose tissue.

We could detect AGMO activity in the murine pre-adipocyte cell line 3T3-L1. Enzyme activity was determined by HPLC based on the conversion of 1-O-pyrenedecyl sn-glycerol to 1-pyrenedecanoic acid [2].To investigate the role of AGMO in the differentiation process from pre-adipocytes to mature adipocytes we manipulated AGMO expression. We will show expression datat of adipocyte markers, AGMO enzyme activity and Oil Red O staining of lipid droplets in mature adipocytes.

[1] Watschinger K, et al. Proc Natl Acad Sci (USA) 2010;107:13672-7.

[2] Werner ER, et al. J Lipid Res. 2007;48:1422-7.

Linking body and mind: A psychoneuroimmunological study of pteridine metabolism and world-view

Schnell T, Hefti R, Schubert C, Fuchs D

Department of Psychology, Leopold Franzens University, and Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria; and Research Institute for Spirituality and Health, Langenthal, Switzerland

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Associations between biological parameters and dimensions of worldview, such as religion and meaning in life, have repeatedly been established. Psychoneuroimmunological processes are not yet clearly understood. To contribute to the clarification of links between worldview and biological functioning, a questionnaire-plus-experimental study has been carried out. A sample of N = 50 persons completed online questionnaires and participated in the Trier Social Stress Test for Groups (TSST-G) (1). Sixty-four percent of the sample were female; mean age was 23 years (SD = 4). The TSST is a procedure to induce stress by means of two socially evaluative situations: the first is a public speaking task framed as part of a job interview; the second is a mental arithmetic task. Both take place in front of a jury of two, as well as up to five other participants; performances are also (mock) videotaped. Saliva samples were taken at baseline, after stressor 1 and 2 and four times during the following recovery period. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the amount of neopterin in saliva.

The mean neopterin level during baseline was 5.26 nmol/L (SD = 3.10); values (and standard deviations) decreased over the course of the stress tasks and reached a stable low value during recovery (4.53/1.3, 4.13/1.22, 3.47/0.86, 3.52/0.72, 3.37/0.65, 3.48/0.87; F(6,44) = 11.42, p <0.001, η2 = 0.61). Women had significantly higher levels of baseline neopterin than men (5.96/3.69 vs. 4.02/0.62). Controlling for sex, baseline neopterin was positively correlated with crisis of meaning (r = 0.27; p <0.05). A negative correlation was established between baseline neopterin and acceptance of existential self-responsibility (r = -0.28, p <0.05). Mean neopterin values were not significantly correlated with worldview dimensions.

Results tie in with previous findings of positive correlations between existential conflict and interleukin-6 [2], and negative correlations between meaning in life and pro-inflammatory interleukin-6 and daily saliva cortisol [3].

[1] Von Dawans A, et al. Psychoneuroendocrinology 2011;36(4):514-22.

[2] Ai A, et al. Psych Rel Spir 2009;1(2):112128.

[3] Ryff, C, et al. Phil Trans R Soc Lond B 2004;354(1449):1383-94.

12 hour-to-12 hour cause-effect relations between IL-6 concentrations and specific and unspecific symptoms in a patient with mild SLE disease activity: A time-series analysis approach

Schubert C, Singer M, Ocaña-Peinado FM, König P, Sepp N, Schnapka-Köpf M, Fuchs D

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

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Little is known about the real-time cause-effect relations between IL-6 concentrations and SLE symptoms. A 52-year old woman with mild SLE activity collected her entire urine for the determination of IL-6/creatinine and protein/creatinine levels (ELISA, HPLC) for a period of 56 days in 12h-intervals. Additionally, she answered questionnaires (VAS) on fatigue, weakness, joint pain, oral ulceration and facial rash, and measured her oral temperature twice a day. Time series analyses consisted of ARIMA modeling and cross-correlational analyses (one lag = 12h, significance level = p <0.05).

Statistical analyses showed that increased urinary IL-6 concentrations preceded increased urinary protein by 36–48h (lag3: r = +0.225) and that, in opposite direction of effect, increased urinary protein preceded urinary IL-6 decreases by 12–24h (lag1: r = –0.322). Moreover, urinary IL-6 increases co-occurred with increased oral ulceration (lag0: r=+,186) and tendentially preceded decreased oral ulceration by 48–60h (lag4: r = –0.170; n.s.). Also, urinary IL-6 increases co-occurred with decreased fatigue (lag0: r = –0.193) and facial rash preceded decreases in urinary IL-6 by 84–96h (lag7: r = –0.215). Body temperature, weakness and joint pain did not significantly correlate with urinary IL-6 concentrations.

Some of the results of this study refer to the existence of real-life feedback mechanisms in SLE. These findings are preliminary and require replication to draw firm conclusions about the real-time association between IL-6 and SLE disease activity.

Routine performance of an “in house” testosterone LC-MS/MS assay: data from the first 500+ days

Seger C

Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), University Hospital, Innsbruck, Austria

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Compared to IVD-CE certified automated ligand binding assays testosterone routine quantification by LC-MS/MS can still be considered a highly experimental procedure which typically relies almost exclusively on laboratory developed measurement procedures. Only a limited number of IVD-CE certified assays are currently available; none of the vendors provides a complete IVD-CE certified instrument / assay solution. Consequently outmost care must be taken by individual laboratories to ensure the analytical integrity of a lab developed or an incompletely certified assay over prolonged times. Preferably calibration systems based on traceable higher order reference materials are utilized, participation in international quality assessment platforms with strong peer groups (e.g. UK-NEQAS) to review calibration preparation accuracy and long-time assay stability are highly recommended.

With the lab developed ZIMCL assay, a online-SPE-HPLC-ESI-MS/MS based testosterone quantification platform approx. 15000 samples have been quantified within the past 18 months. Multilevel calibration is based on materials measured against ERM DA345a and DA346a. Assay accuracy is tested against the UK-NEQAS steroid hormone proficiency testing scheme (72 samples / year). Here the assay was found to be accurate (mean deviation in the LCMS group <1.3%) and precise (standard deviation of this mean 8.8%). The quantification level was set to 0.10 ng/ml which allowed to measure trough almost the complete female reference range – an impossible undertaking if ligand binding assays are utilized.

Schizophrenia and the immune system: possible pathophysiological interactions – an overview

Sperner-Unterweger B, Fuchs D

Department of Psychiatry and Psychotherapy, Division of Psychosomatic Medicine;and Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

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Based on genetic vulnerability, exposure to different kinds of infections and immune activation e.g. due to stress interaction leads to dysbalance of the immune system. Increased inflammatory processes in the periphery as well as in CNS may cause neurobiological changes including changed neurotransmitter metabolisms which result in neurodevelopmental/neurodegenerative alterations yielding to the heterogenous clinical symptoms of schizophrenia.

Analyses of interleukin-6 (IL-6) and kynurenine (KYN) and kynurenic acid- both downstream metabolites of tryptophan – in cerebral spinal fluid of schizophrenia patients indicated that IL-6 interferes with the KYN pathway [1]. These findings are in line with earlier studies which point to a shift in the kynurenic pathway towards enhanced KYNA formation playing a role in the pathophysiology of schizophrenia. Recent results support earlier conclusions drawn from low or even decreased concentrations of macrophage product and pteridine derivative neopterin, on a role of Th2-type immune activation rather than Th1-type immunity in the majority of schizophrenia patients [2, 3]. Also the elevated phenylalanine concentrations and phenylalanine to tyrosine ratios in patients with schizophrenia [4] point towards a role of impaired phenylalanine hydroxylase (PAH) activity and probably tetrahydropteridine (BH4) cofactor availability in the pathogenesis of schizophrenia.

Immune alterations in schizophrenia may reflect underlying pathophysiological mechanisms at least for a subgroup of schizophrenia patients which might be used to a better diagnostic characterization of this heterogenous disease and on the other side these abnormalities may offer further development options of immune related therapeutic strategies

[1] Schwieler L, et al. J Psychiatry Neurosci 2014; 39:140126. doi: 10.1503/jpn.140126.

[2] Sperner-Unterweger B, et al. Schizophr Res 1989; 2:417-21.

[3] Bechter K, et al. J Psychiatr Res 2010; 44:321-30.

[4] Okusaga O, et al. PLoS ONE 2014; 9:e85945.

Bright versus dim ambient light affects subjective well-being but not serotonin-related biological factors

Stemer B, Melmer A, Fuchs D, Ebenbichler C, Kemmler G, Deisenhammer EA

Department of General and Social Psychiatry, Center of Psychiatry and Psychotherapy, Department of Internal Medicine I, and Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

Light falling on the retina is converted into an electrical signal which stimulates serotonin synthesis. Previous studies described an increase of plasma and CNS serotonin levels after bright light exposure. Ghrelin and leptin are peptide hormones which are involved in the regulation of hunger/satiety and are related to serotonin. Neopterin and kynurenine are immunological markers which are also linked to serotonin biosynthesis. In this study, 29 healthy male volunteers were exposed to bright (5000 lux) and dim (50 lux) light conditions for 120 minutes in a cross-over manner. Subjective well-being and hunger as well as various serotonin associated plasma factors were assessed before and after light exposure. Subjective well-being showed a small increase under bright light and a small decrease under dim light, resulting in a significant interaction between light condition and time. Ghrelin concentrations increased significantly under both light conditions, but there was no interaction between light and time. Correspondingly, leptin decreased significantly under both light conditions. Hunger increased significantly with no light-time interaction. We also found a significant decrease of neopterin, tryptophan and tyrosine levels, but no interaction between light and time. In conclusion, ambient light was affecting subjective well-being rather than serotonin associated biological factors.

The influence of bioactive lipid mediators on cholesterol metabolism in sepsis

Tancevski I, Auer K, Asshoff M, Bischof D, Hilbe R, Demetz E, Tymoszuk P, Weiss G

Medical University of Innsbruck, Department of Internal Medicine VI, Infectious Diseases, Immunology, Innsbruck, Austria

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Sepsis is a systemic inflammatory condition following severe bacterial infection resulting in high mortality. Sepsis is a global health problem with an increasing incidence over the past years, accounting for >700,000 cases/year in the United States. During the past years, much has been discovered about the uncontrolled, overwhelming inflammatory condition taking place in an organism affected by sepsis. However, one observation in septicaemic patients still remains enigmatic: why plasma levels of high-density lipoprotein cholesterol (HDL-C) decrease, followed by a decrease in low-density lipoprotein cholesterol (LDL-C). We recently identified a conserved interplay between bioactive lipid mediators including leukotrienes (LTs) and lipoxins (LXs), and HDL-C and LDL-C metabolism [1]. These lipid mediators are typically released by highly specialized white blood cells (neutrophils and monocytes/macrophages) during infection, thereby contributing to inflammation and resolution of infection. During the acute response of innate immunity to bacterial infection mainly neutrophils are attracted to sites of infection with the aim of bacterial killing. These activated neutrophils in turn release cytokines, interleukins, and LTs to attract further neutrophils. After 12-24 hours, neutrophils themselves begin to release so-called stop signals including LXs to slow down the chemoattraction of leukocytes, and to initiate resolution of infection which includes activation of monocytes/macrophages. Our recent studies suggest that sequential generation of these lipid mediators by different leukocytes may lead to critical alterations of HDL- and LDL-receptor expression in the liver, explaining the typical changes in these lipoproteins during sepsis. Here we show a systematic analysis of spatiotemporal generation of lipid mediators combined with changes in cholesterol metabolism in an animal model of Gram-negative sepsis. In summary, our studies will help to further understand the physiology underlying the cholesterol-lowering mechanisms observed in sepsis, placing the proposed thesis at the forefront of immunometabolism research.

[1] Demetz E, et al. Cell Metab 2014;20:787-98.

Macrophages and breast cancer: an iron-hard relationship

Tymoszuk P, Aßhoff M, Doppler W, Weiss G, Theurl I

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

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Tumor-associated macrophages (TAMs) are the major population of tumor infiltrating leukocytes in a plethora of human and animal malignancies whose presence strongly correlates with faster tumor progression and metastatic dissemination. The mechanisms which account for the carcinogenic properties of TAMs are plenty; promotion of angiogenesis, suppression of anti-tumor immunity and growth factor supply are best described.

Clinical and animal studies on breast cancer provide evidence for crucial role of iron in growth of malignant cells and orchestration of tumor-promoting inflammation. In non-malignant tissues as well as in the whole organism macrophages perform the recycling of aged red blood cells and hence regulate iron supply for metabolically active and proliferating cells. The analogical function of TAMs as iron providers for malignant cells attracted, however, only little attention so far. Here, we demonstrate that TAMs present in murine mammary cancer express a broad spectrum of iron turnover proteins and are the sole tumor cell population donating iron to neoplastic epithelium. Furthermore, TAMs, in a close analogy to professional erythrophagocytes of the liver and spleen, ingest aged or deteriorated red blood cells and hence contribute to tumor-promoting effects of blood transfusions.

Summarizing, our findings shed new light on TAM biology by stressing their function in iron turnover. This previously unrecognized aspect has potentially broad clinical implications for the safety of treatment of tumor-accompanying anemia.

Alkylglycerol monooxygenase in murine macrophages

Watschinger K, Keller MA, McNeill E, Alam MT, Laid S, Sailer S, Rauch V, Patel J, Hermetter A, Golderer G, Geley S, Werner-Felmayer G, Plumb RS, Astarita G, Ralser M, Channon KM, Werner ER

Divisions of Biological Chemistry and Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria; Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK; Division of Cardiovascular Medicine, University of Oxford, Oxford, UK; Waters Corporation, Milford, USA; Institute of Biochemistry, Graz University of Technology, Graz, Austria

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Tetrahydrobiopterin-dependent alkylglycerol monooxygenase is essential for irreversible ether lipid cleavage. To investigate the impact of alkylglycerol monooxygenase expression and tetrahydrobiopterin biosynthesis we analysed the lipidome of RAW264.7 with modulated enzymatic activities by liquid chromatography/mass spectrometry. We could identify more than 1000 lipid species, clustered them according to LipidMaps categories/classes/subclasses and performed enrichment analyses. Profound changes in different lipid species beyond the class of ether lipids were revealed and thus, our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in the ether lipid metabolism of murine macrophages.

Supported by the Austrian Science Funds (FWF) [P22406, J3264] and by the autonomous province Bozen-Südtirol, division educational support, university and research.

Ether lipids, metabolism and functional roles

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

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

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Our interest in ether lipids has been initiated by the tetrahydrobiopterin-dependent irreversible degradation of these compounds by alkylglycerol monooxygenase. Alkylglycerols are lipids characterized by the attachment of a fatty alcohol by an ether linkage to the glycerol backbone which is the basis of their designation as ether lipids. The ether bond distinguishes them from the more common glycerol based lipids which have the fatty acid side chains attached by an ester bond. Ether lipids are as abundant and as diverse as their ester counterparts, but the ether bond makes them chemically and metabolically more stable. A subclass of these ether-bound lipids has a double bond adjacent to the ether bond. These vinyl ethers occur mostly as 1-alkenyl-2-acyl-sn-glycero-phospholipids, the plasmalogens, which are the most abundant lipid species in mammalian brain. The vinyl ether bond makes these lipids sensitive to oxidation and acid cleavage.

The biosynthesis of ether lipids is initiated by two key enzymes present in peroxisomes. If one of these two biosynthetic enzymes is inactivated in mice this leads to cataract, male sterility due to the lack of seminolipid, and to alterations in brain structure development. In humans, lack of the first two biosynthetic enzymes in ether lipid biosynthesis leads to severe inherited disease called rhizomelic chondrodysplasia punctata types 2 and 3, respectively. These diseases are associated with cataract, developmental disorders of bone, joints and brain. This results in severe mental retardation and a life expectancy of currently about 10 years. Several enzymes in the ether lipid metabolic pathway have not been characterized by their sequence yet, such as the delta-1 desaturase introducing the vinyl ether bond to form the plasmalogens.

A well characterized biologically active ether lipid mediator is platelet activating factor, a pleiotropic inflammatory messenger. Effects of exogenously added submicromolar concentrations of ether lipids described in the literature include stimulation of macrophages, alteration of the cytokine profiles of T-lymphocytes, inhibition of tumour growth and impact on phospho-signalling pathways, e.g. by inhibition of protein kinase C.

Urinary neopterin and prognosis of patients with gastrointestinal stromal tumors

Zezulová M, Bartoušková M, Študentová H, Kujovská Krčmová L, Solichová D, Melichar B

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

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Increased concentrations of neopterin in serum or urine have been reported in patients with tumors across the spectrum of different primary locations, but there are no reports on neopterin in patients with gastrointestinal stromal tumors (GIST). We have studied urinary neopterin in 51 patients with metastatic GIST. Urinary neopterin was determined by high performance liquid chromatography Increased urinary neopterin concentrations were observed in a significant proportion of patients with metastatic GIST. No statistically significant changes of urinary neopterin were observed during the therapy with imatinib, but a marked decrease was observed occasionally during the first days of imatinib treatment. Increased urinary neopterin did not predict poor survival. In conclusion, urinary neopterin is increased in patients with metastatic GIST. No significant changes were observed in association with imatinib therapy. Increased urinary neopterin was not associated with inferior survival.

About the article

Published Online: 2015-06-20

Published in Print: 2015-09-01

Citation Information: Pteridines, Volume 26, Issue 3, Pages 113–133, ISSN (Online) 2195-4720, ISSN (Print) 0933-4807, DOI: https://doi.org/10.1515/pterid-2015-0007.

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