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Pteridines

Official Journal of the International Society of Pteridinology

Editor-in-Chief: Fuchs, Dietmar


IMPACT FACTOR 2018: 0.531

CiteScore 2018: 0.67

SCImago Journal Rank (SJR) 2018: 0.195
Source Normalized Impact per Paper (SNIP) 2018: 0.318

ICV 2018: 145.86

Open Access
Online
ISSN
2195-4720
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Volume 28, Issue 1

Issues

36th International Winter Workshop Clinical, Chemical and Biochemical Aspects of Pteridines and Related Topics

Published Online: 2017-04-20 | DOI: https://doi.org/10.1515/pterid-2017-0006

*)These abstracts have been reproduced directly from the material supplied by the authors. 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

and

The Austrian Society of Laboratory Medicine and Clinical Chemistry

Held in Innsbruck, Tyrol, Austria

February 21st–24th, 2017

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

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

Association between neopterin and citrulline, a biomarker of gut function, during treatment with nivolumab

Bartoušková M, Spisarová M, Vitásková D, Študentová H, Hudcová M, Adam T, Melichar B

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

( )

Although the gastrointestinal toxicity is one of the principal side effects of systemic therapy, the assessment still relies mostly on the symptoms reported by the patient. In a prior study in patients with rectal cancer we have demonstrated that citrulline represents a promising biomarker of gastrointestinal toxicity. In an earlier study, we have demonstrated a decrease of circulating citrulline levels accompanied by an increase in urinary neopterin, a biomarker of immune response, in rectal cancer patients treated with chemoradiation. Both citrulline and neopterin concentrations correlated with the radiation dose. The advent of immune checkpoint inhibitors, e.g. ipilimumab or nivolumab, has virtually transformed the management of a number of solid tumors. However, the search for reliable predictive biomarkers of immunotherapy continues. In the present study, citrulline and neopterin concentrations were studied in non-small cell lung cancer and renal cell carcinoma patients treated with nivolumab. First preliminary data from the pilot study that investigated plasma citrulline, serum C-reactive protein (CRP) and neopterin, and peripheral-blood cell count (PBC)-derived ratios (that represent relative lymphocyte counts), are presented here. At baseline as well as during the treatment both urinary neopterin and CRP significantly correlated with PBC-derived ratios. Among the biomarkers investigated only serum neopterin was consistently increased throughout the course of nivolumab therapy compared to baseline concentrations. Notably, no decrease in plasma citrulline was observed, corresponding to a good tolerability of the treatment. In conclusion, preliminary data indicate that the activation of the immune response associated with nivolumab therapy results in significantly increased circulating neopterin concentrations. Both CRP and serum neopterin correlates with PBC-derived ratios in this patient population treated with immunotherapy. The collection of data from additional patients and the data analysis are continuing.

Toxoplasma breaks, lithium fixes: does dopamine connect aggression, impulsivity, and suicide effects of toxoplasmosis

Can A, Postolache T

Department of Psychiatry, University of Maryland School of Medicine, Baltimore MD, USA

( )

Behavioral effects of T. gondii infection manifest themselves as increased risk taking, impulsivity, aggression in both humans, and animal models. Suicide risk is also heightened in individuals with toxoplasma seropositivity. Previous research showed that rate limiting enzyme of dopamine synthesis, tyrosine hydroxylase is expressed in T. gondii bradyzoites and found in high concentrations in tissue cysts. Further, blood levels of dopamine precursors phenylalanine and tyrosine are modified by low grade inflammation, and have been recently identified as a moderator of the association between T. gondii infection, and trait impulsivity and aggression. Lithium is a mood stabilizer that reduces mania like behavioral phenotypes in humans and rodents, including aggression and impulsivity. Moreover, chronic lithium is the most effective treatment of suicidal behavior. This significant overlap between the behavioral effects of toxoplasmosis and therapeutic effects of lithium suggest that they may share common neurobiological mechanisms through their effects on the dopaminergic system. We tested whether lithium treatment affected dopaminergic transmission in the brain. We utilized fast-scan cyclic voltammetry which allows near real-time changes in dopaminergic concentrations to monitor changes in extracellular DA concentrations in the nucleus accumbens (NAc) core with acute or chronic lithium treatment in a mouse model. We also measured electrically induced dopamine release after amphetamine treatment, which modeled mania-like phenotype. Chronic, but not acute, lithium treatment attenuated dopamine release under both conditions. These results showed that lithium’s anti-manic effects may be mediated by its effect on the dopaminergic signaling. Taken together, we propose that lithium may reduce, at least in part, aggression, impulsivity and overall risk taking previously linked with T. gondii infection symptoms through a dopaminergic mechanism.

Establishment of a 3D respiratory model to study airborne infections

Chandorkar P, Posch W, Blatzer M, Ammmann C, Lass-Flörl C, Wilflingseder D

Division of Hygiene and Medical Microbiology, Department of Experimental Orthopaedics, Department of Anaesthesiology and Critical Care Medicine, Medical University Innsbruck, Austria

( )

Aspergillus (A.) fumigatus causes life-threatening pulmonary infections in patients with a challanged immune system. Current studies to better understand the fungal invasion process are primarily performed in animal models and cell lines, both afflicted with drawbacks. Thus, we set up a perfused 3-dimensional in vitro cell culture model with primary, differentiated human bronchial epithelial cells and immune cells important to sense fungal pathogens, i.e. alveolar macrophages or dendritic cells (DCs). Our highly sophisticated cell culture model is suited to study fungal-epithelial-immune interactions at the entry sites of the pathogen. Development, differentiation and interactions with A. fumigatus of epithelial - immune cell co-cultures under static or perfused conditions were studied using confocal, scanning electron and live cell microscopy. Cytokine analyses from Aspergillus -exposed tissues and TEER measurements were performed. Analysis over time by confocal and live cell microscopy showed that respiratory cells differentiated in an air-liquid interface to form tight junctions, produced mucus and developed cilia - this process was significantly accelerated under perfused compared to static conditions. Upon fungal infection, A.fumigatus was trapped in the muco-ciliary layer up to 3h before internalization by epithelial cells. In epithelial-immune cell co-cultures, migration of DCs from basolateral to apical side was detected upon fungal infection, where DCs co-localized with fungal hyphae. Our model will provide novel immunologic and mechanistic insights into Aspergillus-infection processes within 3D space. Furthermore, this model provides a vast array of applications with respect to respiratory challenges and diseases and can be exploited in terms of translational goals. Additionally, animal experimentation can be significantly reduced by use of this highly developed human system, thereby contributing to ethical considerations and higher biological relevance in terms of avoiding interspecies differences.

Novel redox roles of tetrahydrobiopterin

Channon KM

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

( )

Further reading:

Chuaiphichai S, Crabtree MJ, Mcneill E, Hale AB, Trelfa L, Channon KM, Douglas G. A key role for tetrahydrobiopterin-dependent endothelial NOS regulation in resistance arteries: studies in endothelial cell tetrahydrobiopterin-deficient mice. Br J Pharmacol 2017 Jan 27. doi: 10.1111/bph.13728.

Bailey J, Shaw A, Fischer R, Ryan BJ, Kessler BM, McCullagh J, Wade-Martins R, Channon KM, Crabtree MJ. A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance. Free Radic Biol Med 2017 Mar;104:214-225. doi: 10.1016/j. freeradbiomed.2017.01.012.

(Abstract not received in time for inclusion)

A positive association between Toxoplasma gondii IgG serointensity and current hopelessness-dysphoria scores in the Old Order Amish: a preliminary study

Dagdag AT, Wadhawan A, Daue LM, Peng X, Bazaco MC, Markon AO, Punzalan CM, Groer MW

Postolache TT Mood and Anxiety Program, Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, USA; Adult Psychiatry, University of Maryland Medical Center, Baltimore, MD,USA; Program for Personalized and Genomic Medicine, Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA; US Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Analytics and Outreach, Division of Health Informatics and Analytics, Epidemiology Branch, College Park, MD,USA; University of South Florida, Tampa Fl, USA; The VA Rocky Mountain MIRECC for Suicide Prevention, Denver, CO, The VISN 5 MIRECC, Baltimore, MD

( )

Toxoplasma gondii (T.gondii) IgG positivity and serointensity, and markers of low-grade inflammation, have been previously associated with suicidal self-directed violence (SSDV). Although associations with unipolar depression have also been investigated, results have been inconsistent, possibly as a consequence of high heterogeneity of clinical samples. Thus, we now studied in a more homogeneous population, (i.e. Old Order Amish) with high T.gondii seroprevalence >50%, associations between markers of T.gondii infection and hopelessness-dysphoria and anhedonia scores on depression screening questionnaires. As individual symptoms of depression (such as hopelessness) carry an increased risk for SSDV, we now analyze for the first time, to our knowledge, individual associations of anhedonia and hopelessness/dysphoria.

In 306 Old Order Amish, age range 18-87 (median 44), with 191 (62.4%) women, all participants in the Amish Wellness Program in Lancaster, PA, we had obtained T.gondii IgG-titers and neopterin levels (by ELISA), and depression screening questionnaires (based on PHQ9 N=280 and PHQ2). Current and life-long anhedonia, hopelessness-dysphoria, combined anhedonia and hopelessness-dysphoria, and either anhedonia or hopelessness-dysphoria scores were analyzed in relationship to log-transformed titers, seropositivity, with multivariate linear models with adjustment for age and sex.

Serointensity was significantly associated with current hopelessness-dysphoria (p = 0.045) and combined anhedonia and hopelessness-dysphoria (0.040), while associations with simple anhedonia scores, life-long depression phenotypes, were not significant. There was also a marginal trend for associations between seropositivity and current hopelessness-dysphoria (p = 0.057) and their combination (p = 0.055). These results will first be replicated in a larger sample allowing additional adjustments for covariates and multiple comparisons, and if remaining significant, may lead to prospective longitudinal studies that could test if exacerbation in hopelessness-dysphoria score is associated with IgG-titers elevations, possibly secondary toT.gondii reactivation. Additional analyses of serotype, toxoplasma infection risk factors, retina markers of infection and brain imaging are also planned. Current hopelessness being a known risk factor for SSDV, and responding to treatments- such as cognitive behavioral therapy, it might clinically relevant to monitor and target hopelessness in T. gondii positive individuals at increased risk for suicide.

This study was supported by Distinguished Investigator Award from the American Foundation for Suicide Prevention (DIG 1-162-12, T. Postolache); P30 DK072488 NIDDK (NORC pilot/developmental grant PI from the NIDDK, NIH, Bethesda, MD; the Joint Institute for Food Safety and Applied Nutrition, and the US FDA, through the cooperative agreement FDU.001418 (T. Postolache); and the MHBA-016-15S, Merit Award from VA CSR&D (T Postolache). The results and their interpretation are of the authors, and do not represent official views of the VA, FDA or AFSP.

PCBP2: a new jigsaw piece in iron dependent host response

Dichtl S, Hilbe R, Seifert M, Berger S, Lutz O, Nairz M, Weiss G

Department of Internal Medicine VI, Medical University of Innsbruck, and Austrian Drug Screening Institute, Innsbruck, Austria

( )

Intracellular iron chaperons like Poly(rC)-binding proteins (PCBPs) are not very well investigated in mammalians until now. PCBPs are involved in intracellular iron shuttling. The major isoforms PCBP1 and 2 bind iron with low micromolar affinity and deliver it to proteins and enzymes but are maybe also involved in RNA binding and translational control of gene expression. Thereby, PCBP1 or 2 may affect the expression of the iron transporters DMT1 or ferroportin and the iron storage protein ferritin. Based on lots of open questions regarding the role of PCBPs in macrophages, we investigate the effect of PCBP2 knockout on iron metabolism at base-line and in Salmonella typhimurium (S.tm.) infection.

We developed CRISPR-Cas9 PCBP2 knockout murine J774 macrophages to study the regulation of PCBP2 on iron homeostasis. To find out if PCBP2 is an essential piece in Salmonella dependent host response, we infected the mentioned PCBP2 KO macrophages with the murine pathogenic Salmonella strain Salmonella typhimurium.

A knockout of PCBP2 resulted in a decreased ferroportin level, the major iron exporter, and a higher ferritin expression, which results in an increased intracellular iron content as compared to PCBP2 expressing macrophages. These findings were consistent with an increased uptake of iron and a reduced iron release by PCBP2 depleted cells. To study the consequence of this, in regard to the control of infection with siderophilic intracellular bacteria, we infected wt and PCBP2 depleted macrophages with Salmonella typhimurium. PCBP2 depletion resulted in an increased intracellular proliferation of Salmonella and reduced infection control. In parallel PCBP2 depletion also impaired anti-microbial immune responses compared to wt macrophages as reflected by an increased expression of IL-6.

Our data demonstrate that PCBP2 is crucial for the control of iron metabolism in macrophages. In Salmonella infection, PCBP2 contributes to host defense by strengthening antimicrobial immune defenses and reducing iron availability for intracellular bacteria. Therefore PCBP2 appears to be an attractive therapeutic target for infections in an era of increasing antimicrobial resistance.

Effect of KynA on N-methyl-D aspartate receptor-coupled glucose transporter-3 in SH-SY5Y dopaminergic neurons exposed to high glucose

Engin AB, Engin ED, Aral A, Gülbahar Ö, Engin A

Gazi University, Faculty of Pharmacy, Department of Toxicology, Hipodrom,; Ankara University, Biotechnology Institute, Tandogan; and Gazi University, Faculty of Medicine, Department of Immunology, Department of Biochemistry, Besevler, and Department of General Surgery, Besevler, Ankara, Turkey

( )

Unlike peripheral resistance, brain insulin resistance does not affect classic glucose uptake. However, the “insulin-resistant brain state” has been hypothesized to form the core of the neurodegenerative events that occur in sporadic Alzheimer’s disease. In patients with neurodegenerative diseases, in addition to abnormalities in neuronal glucose metabolism, the levels of glucose transporter-3 (GLUT3), the major brain glucose transporter, is decreased. The regulation of glucose transport is achieved by regulatory induction of the surface expression of GLUT3. Previously, the role of N-Methyl-D Aspartate receptor (NMDAR) in the surface expression of GLUT3 was studied by an NMDAR open-channel blocker, MK801 (dizocilpine maleate). However, non-competitive NMDAR antagonist, MK-801 induces necrotic cell death in neurons by oxidative stress due to increase in glucose metabolism in many brain areas. Therefore, the mechanism by which neuronal activity controls glucose influx through GLUT3 expression by an endogenous specific NMDAR antagonist, kynurenic acid (KynA), was studied. SH-SY5Y neuroblastoma cells were exposed to 40 uU/mL insulin and (150-250 mg/dL) high glucose considering International Diabetes Federation criteria. In KynA and neuronal nitric oxide synthase inhibitor, L-NAME supplemented culture media, oxidative stress, total mitochondrial metabolic activity/cell viability percentage (MTT), nitric oxide and GLUT3 were determined at 24 and 48 h incubation periods. Viable cells were counted by trypan blue dye. Total mitochondrial metabolic activity of the high glucose-exposed SH-SY5Y cells were decreased, while the oxidative stress increased. These cells showed two times more GLUT3 expression in comparison to controls. GLUT3-stimulated glucose transport and glycolysis-induced oxidative stress were enhanced, but simultaneously the total mitochondrial metabolic activity was reduced. Insulin supplementation to high glucose-containing medium caused decreases in NMDAR-mediated nitric oxide synthesis and GLUT3 activation. Eventual decrease in glucose entry into cells significantly reduced cell viability by stimulating oxidative stress. In high glucose-exposed SH-SY5Y cells, KynA significantly reduced oxidative stress and increased cell viability by tightly regulating NO production and GLUT3 expression. KynA significantly prevented neuronal damage in high glucose-exposed SH-SY5Y cells. KynA a noteworthy compound with important advantages, as it is endogenously synthesized, specific NMDAR antagonist and significantly reduces oxidative stress.

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

Immunotoxicity of formaldehyde and formaldehydogenic compounds

Fagundes dos Santos P, Stonig M, Warter A, Geisler S, Fuchs D, Gostner JM

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

( )

Several aspects of formaldehyde toxicology have been investigated in depth over the past decades. However, the information about its effect on the human immune system is still sparse [1]. Due to the complexity of the immune system, it is of importance to apply reliable biomarkers that may specify an immunologic effect of a chemical. The activity of the immunoregulatory enzyme indoleamine 2,3-dioxygenase-1 (IDO-1) was shown to be an indicator of either immune-activating or immunosuppressive responses in a variety of studies investigating the effects of compounds [2,3]. In this study, the effect of formaldehyde and of the formaldehydogenic compounds methanol and aspartame were investigated in the in vitro model of human peripheral blood mononuclear cells (PBMC) that were stimulated or not with a mitogen [3]. IDO-1 is induced upon stimulation and converts the essential amino acid tryptophan to kynurenine. This is the first and rate limiting step of the kynurenine breakdown pathway leading to manifold immunological consequences.

The activity of IDO-1, as indicated by the kynurenine to tryptophan ratio, was significantly suppressed in stimulated cells upon formaldehyde addition at low micromolar concentrations. Physiologically occuring formaldehyde concentrations in human blood are in the same order of magnitude. A half maximal inhibitory concentration (IC50) of approximately 50 μM was calculated for IDO-1 suppression. Similar effects could be observed in unstimulated cells, however at higher concentrations only (IC50 > 200 μM). Treatments with 100 μM formaldehyde already decreased cell viability significantly with only slight differences in the response of unstimulated compared to stimulated cells. However in stimulated cells, the suppressive effect of formaldehyde on IDO-1 was already detectable at concentrations where the cell viability was only slightly reduced. Thus, formaldehyde inhibition of tryptophan breakdown may be an early marker of cytotoxicity.

At the cell surface, formaldehyde may covalently bind to amine and thiol groups of macromolecules and it is yet not clear to which amount it is taken up into the cells, where it is metabolized to formate. Similar concentrations of formate did not affect IDO-1 activity in the PBMC model. The formaldehydogenic compounds methanol and aspartame were able to suppress IDO-1 activity, however these effects were observed at the higher micromolar to millimolar concentration range only. In general, the effects were stronger in stimulated cells.

Data showed that tryptophan metabolism is affected by formaldehyde. It can be suggested that this is a combined effect deriving from of extracellular and intracellular events. IDO-1 suppression was more pronounced in an inflammatory environment. It is likely that at low concentration the antioxidative capacity of formaldehyde favors a reductive milieu, thereby suppressing Th1-type related immunobiochemical pathways such as IDO-1 activity. A similar trend was observed in occupational studies [4,5]. Whether the electrophilic properties of formaldehyde will activate stress pathways that propagate adverse reactions in this immunosuppressed environment needs further investigations.

[1] Veraldi A, et al. Am J Ind Med 2006;49:1046-55.

[2] Gostner JM, et al. Immunol Lett 2015;168:285-92.

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

[4] Jia X, PLoS One 2014;9(8):e104069.

[5] Seow WJ, Carcinogenesis 2015;36:852-7.

Tryptophan metabolism as a key element in partner seeking: a hypothesis

Fuchs D, Geisler S, Gostner JM

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

( )

Throughout decades researchers try to understand the relationship between various aspects of human biology in regard to interpersonal attraction and mate choice. The majority of information about relevant biochemical mechanisms originates from animal studies. A central role was attributed to the major histocompatibility complex (MHC), as a certain degree of MHC protein diversity is important for immunocompetence. The MHC is suggested to be a major genetic factor involved in mate choice by transporting information about genetic relationship and individuality [1]. A relationship of MHC composition and an individual’s odour is discussed, since olfaction is a central element for social communication in vertebrates [2]. In addition, MHC diversity appears to be a determining factor for successful pregnancy along with other components of the immune system. At this, tolerance inducing pathways such as tryptophan metabolism via the enzyme indoleamine 2,3-dioxygenase-1 (IDO-1) are of importance. It can be suggested that very discrepant MHC composition of parents will evoke a more intensive immune response thereby stimulating IDO-1 activity, which promotes the establishment of an immunosuppressed and tolerant environment around the implanting embryo, enhancing the success rate for completion of pregnancy.

Aside from immunobiological perspectives, an important component in partner selection is certainly sympathy in which a positive attitude, happiness and smiling habit are of greater importance. There are some discrepancies in the definition of happiness in the field of positive psychology, depending on whether it is discussed as a state in emotional terms or as general life-satisfaction or subjective well-being [3]. Also social and cultural aspects play a role. However, signs of a bad mood are usually not found to be very attractive. It seems that humans prefer to be surrounded by smiling faces and this is particularly true in partner selection which is intended to bring partners together for a prolonged life span. In addition, there are several studies that happy individuals are successful across multiple life domains including personal relations and health, arguing for a happiness-success link [4].

The question arises why a smiling face is so attractive, and the answer goes probably far beyond the fact that happy and humorous people are less boring than the others. Biochemically, driving forces of happiness are specific neurotransmitters and thereby serotonin (5-hydroxytryptamine, 5HT) plays a major role. The biosynthesis of 5HT starts with the essential amino acid tryptophan which is converted by tryptophan 5-hydroxylase to 5-hydroxytryptophan, which is then decarboxylated to 5HT. The availability of tryptophan is a key element for the production of 5HT. However, the largest amount of tryptophan is utilized by the so-called kynurenine pathway, which is crucial for the regulation of Th1-type immunity having IDO-1 as a central player. Due to accelerated IDO-1 activity in various immunopathologies, tryptophan breakdown is enhanced and patients may present with tryptophan concentrations lower than normal. Often, such conditions were reported to be associated with low mood, impaired quality of life and increased mortality risk [5-7].

In summary, associations of low tryptophan and low mood were reported in several settings, and it can be hypothesized that low 5HT availability is one of the factors that may reduce the smile in the face of a victim of enhanced tryptophan breakdown. Though rather simplified, smiling faces may attract partners more because of the more glorious future that can be expected in comparison to a victim of low mood.

[1] Chaix R, et al. Plos Genet 2008 4: e1000184.

[2] Hinz C, et al. Sci Rep. 2013 30;3:2800.

[3] Pileggi Pawelski S. Scientific American Mind 2011; 22:50-55.

[4] Lyubomirsky S, et al. Psychol Bull. 2005;131(6):803-55.

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

[6] Murr C, et al. Eur J Clin Invest 2015;45:247-54.

[7] Weinlich G, et al. Dermatology 2007;214:8-14.

Measurements of neopterin and aromatic amino acids in saliva

Geisler S, Gostner JM, Schnell T, Fuchs D

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

( )

Saliva has become an interesting laboratory diagnostic fluid for clinical evaluation and research in the last years, as it can be deduced from increase of recent articles dealing with the diagnostic potential of saliva [1]. Salivary fluid seems to reflect the current physiological condition of the body, which is due to the high permeability of salivary glands and surrounding capillaries. Therefore, there is a steady exchange of molecules between plasma and salivary gland. In response to acute stress, several biological markers of inflammation have already been detected in saliva specimens, such as interleukin-1ß (IL-1ß), IL-6 or tumor necrosis factor-α (TNF-α), but few studies have been conducted on the relation of amino acid levels in saliva during inflammation conditions. One hundred and fifty one saliva specimens were obtained from students and concentrations for tryptophan (Trp), kynurenine (Kyn), tyrosine (Tyr) and phenylalanine (Phe) were determined by advanced HPLC methods as described earlier [2,3]. Saliva neopterin concentrations were measured by ELISA (BRAHMS, Henningsdorf, Berlin). Phe and Tyr concentrations were 73.5 ± 45.7 μmol/L and 89.0 ± 45.2 μmol/L and average neopterin concentrations were 5.2 ± 3.0 nmol/L concentrations being similar to serum levels. However, Trp concentrations were mean ± S.D. 1.48 ± 3.0 μmol/L which is approximately 50-fold lower as compared with serum levels 65 ± 10 [4]. Kyn concentrations were mostly below the HPLC detection limit of 0.1 μmol/L.

The surprisingly low saliva Trp concentrations could be due to several factors like mouth flora, nutrition, dilution or influence on Trp exchange between capillaries and salivary glands. Considering an influence on the Trp exchange, the model of albumin bound (90-95%) and (non-albumin-bound) free Trp (5-10%) species in the plasma [5] would provide an alternative explanation, when it is assumed that only free Trp could easily diffuse from the blood to the salivary gland and is further presented in saliva. Further investigation should be conducted, as e.g. parallel measurements of free Trp in plasma and saliva, using methods as suggested by Badawy like diffusion by avoiding precipitation of proteins which would set free serum-bound albumin [5].

Existing significant correlations between neopterin and saliva amino acids Trp (rs=0.430; p < 0.01), Tyr (rs=0.591; p < 0.01) and Phe (rs=0.495; p < 0.01) obviously do not reflect the plasma relations of the amino acids and neopterin, but indicate additional influencing factors as dilution or local infections. The measurements in saliva may not clearly depict peripheral blood measurements, however in future, the deeper understanding of metabolite exchange processes between saliva and plasma will strengthen the great value of saliva analysis in diagnostics and research.

[1] Slavish DC, et al. Brain Behav Immun 2015;44:253-69.

[2] Neurauter G, et al. Clin Biochem 2013;46(18):1848-51.

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

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

[5] Badawy AA. Neuropharmacology 2017;112(Pt B):248-63.

Evaluation of urinary neopterin levels in children with carbon monoxide poisoning

Girgin G, Sabuncuoğlu S, Tekşam Ö, Özgüneş H, Baydar T

Hacettepe University, Faculty of Pharmacy, Toxicology Department; Hacettepe University, Faculty of Medicine, Department of Pediatrics, Division of Pediatric Emergency, Ankara, Turkey

( )

Carbon monoxide (CO) is a well-known environmental toxicant and the leading cause of morbidity and mortality by poisoning around the world. The affinity of CO for hemoglobin is more than 200-fold greater than the affinity of O2. Carboxy hemoglobin (COHb) values correlate poorly with clinical outcome. Even in mild CO poisoning, delayed neurological sequelae can still occur. Therefore, beyond COHb-mediated hypoxia, additional pathophysiological mechanisms are believed to exist. Clinical management of acute CO poisoning involves supportive care and supplemental oxygen administration. In some cases hyperbaric oxygen treatment may also be considered. It has been reported that, prolonged asphyxia results in increased number of alveolar macrophages and lymphocyte activity. Neopterin is a sensitive marker for the activation of cellular immune system. Its levels in body fluids can be regarded as an indirect estimate of the degree of oxidative stress. The amounts of neopterin produced by activated monocytes/macrophages correlate with their capacity to release reactive oxygen species (ROS). The aim of this study was to evaluate urinary neopterin levels of children who were admitted to emergency service with CO poisoning. 47 children with CO poisoning (24 girls, 23 boys; aged 1-17), and as controls 30 healthy children (ages 2 to 18) were admitted to the study. Urinary neopterin, biopterin, creatinine and serum kynurenine and tryptophan levels were analyzed by HPLC. Serum neopterin levels were determined by ELISA (IBL, Germany). The only significance was observed between COHb levels of CO exposed and control groups. Although serum and urinary neopterin levels and kynurenine to tryptophan ratio were higher in CO exposed children, no statistical significance was observed and the measured parameters did not follow a certain pattern. There were no significant changes in any of the parameters between pre- and post-oxygen treatment of patients. CO can cause oxidative stress, inflammation and immune activation. Most of the data related to CO poisoning is obtained from animal studies, hence human data is important. As the initial clinical presentation does not predict the outcome in CO poisoning, markers predicting clinical outcomes or possible neurological effects would be supportive. Follow-up studies are planned to investigate the effect of hyperbaric oxygen treatment on parameters related to pteridine pathway and to investigate whether neopterin levels or kynurenine pathway components can predict the clinical outcome.

What is all about: neopterin-biopterin-tryptophan in biomedicine

Gostner JM, Geisler S, Fagundes P, Fuchs D

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

( )

The research on the unconjugated pteridine derivative biopterin (2-amino-6-(1,2-dihydroxypropyl)-1H-pteridin-4-one) was intensified after the demonstration of 5,6,7,8-tetrahydrobiopterin (BH4) to be required as a cofactor for aromatic amino acid monooxygenases such as phenylalanine hydroxylase (PAH) by Seymor Kaufmann and his group in 1978 [1]. One year later, an article published by Helmut Wachter et al. shed some light on neopterin, which was present in increased amounts in the urine of patients suffering from virus infections or cancer [2]. Further studies in human peripheral blood mononuclear cells (PBMC) revealed that the activated immune system is a source of increased neopterin levels in humans suffering from diseases involving cell-mediated (Th1-type) immune reactions [3]. Later, the Th1-type cytokine interferon-γ (IFN-γ) was recognized as the key component to stimulate GTP-cyclohydrolase I, which is responsible for the biosynthesis of pteridine species [4]. In human monocyte-derived cells like macrophages or dendritic cells, neopterin is the major product whereas in other cell types and cells including macrophages from other species, its sister compound BH4 is produced primarily instead of neopterin.

The chromatograms obtained by analyzing supernatants collected from stimulated human PBMC showed that the production of neopterin was accompanied by the breakdown of the essential amino acid tryptophan [5]. This finding opened a new understanding about the co-existence of elevated neopterin concentrations with an enhanced tryptophan breakdown rate (increased kynurenine to tryptophan ratio) together with neuropsychiatric symptoms like cognitive impairment, sleep disturbances and depressive mood in various diseases [6]. Finally, the association between increased neopterin and disturbed phenylalanine metabolism directed the research towards a possible influence of reactive oxygen species released during immune responses PAH activity [7]. On the one hand, IFN-γ induced formation of the oxidation-sensitive cofactor BH4 and on the other hand ROS species produced in parallel, interfere with the activity of PAH.

[1] Kaufman S, et al. N Engl J Med 1978;299:673-9.

[2] Wachter et al. Hoppe Seylers Z Physiol Chem 1979;360:1957-60.

[3] Fuchs D, et al. Hoppe Seylers Z Physiol Chem 1982;363:661-4.

[4] HuberC, et al. J Exp Med 1984;160:310-6.

[5] Werner ER, et al. Life Sci 1987;41:273-80.

[6] Widner B, et al. Brain Behav Immunity 2002;16:590-5.

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

Cellular reaction to VOC exposures

Gostner JM, Zeisler J, Alam MT, Martini S, Fagundes dos Santos P, Geisler S, Hermann M, Fuchs D

Division of Medical Biochemistry and Division of Biological Chemistry, Biocenter, and Department of Anaesthesiology and Critical Care Medicine, Medical University of Innsbruck, Bioenergy2020+, Graz, Austria; Warwick Medical School, University of Warwick, Coventry, UK

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In recent years, special attention has been paid to volatile organic compounds (VOC) present in indoor environments. Associated adverse effects are mostly driven by chronic exposure to low concentrations and include respiratory tract irritation and sensitization leading to the development of allergy and asthma.

To investigate cellular reactions that are initiated by low-dose of volatile compounds in an in vitro approach, we developed an exposure platform prototype for airborne treatments. For the first time, this exposure system allows the long-term treatment of air-liquid interface (ALI) lung cell cultures with volatile compounds over a longer period of time [1]. In a proof of principle study ALI cultures of A549 lung cells were exposed to formaldehyde at regulatory threshold concentrations over a period of three days. Despite the low exposure concentrations of 0.1 and 0.5 ppm, differential response patterns emerged in an unbiased functional genomics approach, indicating, e.g., lipid biosynthetic pathways to be affected at lower concentrations, while higher concentrations already indicated changes in proliferation, differentiation and apoptotic signaling pathways, though cell viability was not yet affected. A limitation is certainly that only few molecular interactions, which can be retrieved from databases, are characterized in a low-level perturbation context, where non-monotonic and hormetic responses are important. As the response of epithelial cells to external stimuli is usually moderate due to their barrier function, further research focused on the integration of different immune cell populations in the cell model as this is not only useful to amplify signals but also to extrapolate findings with regard to systemic effects. A major goal is the identification of exposure biomarkers, whereby the tryptophan breakdown pathway via indoleamine 2,3-dioxygenase (IDO-1) is a potent target for the assessment of immunomodulatory effects [2,3].

[1] Gostner JM, Sci Rep 2016 1;6:37842.

[2] Gostner JM, et al. Immunol Lett 2015;168:285-92.

[3] Fallarino F, Cell Cycle 2014;13:2645-6.

On the functional cause-effect-relations between interleukin-6 and mood, irritation and mental activity in a breast cancer survivor

Schubert C, Hagen C

Clinical Department of Medical Psychology, Innsbruck Medical University, Innsbruck, Austria

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The inconsistency in findings on the relationship between emotions and interleukin-6 (IL-6) levels in breast cancer patients may be attributable to the methodological neglect of the topic’s dynamic characteristics. This ‘integrative single-case study’ (Schubert et al., 1999) investigated the bidirectional cause-effect-relations between various emotional states (i.e., mood, irritation, mental activity) and urinary IL-6 levels in a 49-year old female breast cancer survivor under conditions of ‘life as it is lived’. During a period of 28 days the patient collected her entire urine in 12 h intervals for IL-6 measurement and completed every morning and evening a list of adjectives regarding mood, irritation and mental activity (55 measurements in total). ARIMA modeling revealed a 4-day cycle in the IL-6 time series. Furthermore, cross-correlational analyses after controlling for serial dependencies (significance level: p < 0.05) showed that increases in mood and mental activity, as well as decreases in irritation were followed by decreases in urine IL-6 levels with temporal delays between 12 and 36 h. In the opposite direction of effect, increases in urinary IL-6 levels were followed by increases in mood and mental activity, as well as decreases in irritation with temporal delays between 48 and 72 h. These cross-correlational results are strong indicators of real-life negative feedback loops and demonstrate that IL-6 could be involved in health rather than sickness behavior. The study confirms suggestions that considering dynamic characteristics of variables is essential for an adequate understanding of the functional emotion-immune interplay in psychoneuroimmunology (PNI) research.

Signal integration by the intrinsically disordered protein p27Kip1

Hengst L, J ä kel H, Masuccio A, Peschel I, Podmirseg SR, Ranches GD, Roilo M, Vosper J

Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria

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The intrinsically disordered protein (IDP) p27Kip1 was originally identified as a CDK inhibitor protein that binds to and inactivates cyclin-dependent kinases (CDKs). Increasing evidence revealed that this initial characterization oversimplifies its rather complex functions in cell cycle control and other physiological processes. For example, upon modification following mitogen activation, the CDK inhibitor can be transformed into an assembly factor and activator of CDKs. In addition, CDK-independent functions of p27 have been uncovered that include the regulation of cytoskeletal dynamics, cell motility and cell invasion. This has linked p27 to cancer metastasis.

Multiple diverse signals converge at the level of p27, suggesting that the protein serves as a platform for signal integration. They regulate transcription and translation, but also function, stability, localization or protein interactions. Posttranslational modifications include phosphorylation or proteolytic processing. Caspases can induce cleavage of p27 that uncouples its stability from cell cycle regulation and abolishes its ability to stimulate cell migration and invasion.

In addition to their role in programmed cell death, caspases exert non-lethal functions in diverse developmental processes including cell differentiation or tissue remodelling. Activated caspases cause proteolytic processing of p27 and remove a C-terminal fragment of 22 amino acids from the CDK inhibitor, including a phosphodegron. Thereby caspases protect the inhibitor from SCFSkp2-mediated proteasomal degradation in S, G2 and M phases of the cell cycle. As a consequence, p27 becomes stabilized and remains an efficient inhibitor of cell cycle progression. In addition, following processing by caspases, p27 fails to bind to RhoA and to inhibit its activation. This abolishes the ability of p27 to stimulate cell migration and invasion. The stabilization of the CDK inhibitor and elimination of RhoA induced cytoskeletal remodelling upon caspase processing could contribute to cell cycle exit and cytoskeletal remodelling during non-lethal caspase controlled differentiation processes.

Tryptophan metabolism and its relationship with central nervous system toxicity in subjects switching from efavirenz to dolutegravir

Keegan MR, Winston A, Higgs C, Fuchs D, Boasso A, Nelson M

Imperial College London, United Kingdom; ViiV Healthcare Ltd, UK; Chelsea & Westminster Hospital, London, United Kingdom; Innsbruck Medical University, Innsbruck, Austria

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The pathogenesis of central nervous system (CNS) toxicities observed in antiretroviral (ART) treated persons living with HIV (PLWH) remain elusive. We investigated the associations between indoleamine 2,3-dioxygenase-1 (IDO-1) activity, via kynurenine/tryptophan (KYN/TRP) ratios, and measurements of CNS toxicity in PLWH switching from efavirenz (EFV) to dolutegravir (DTG).

In a prospective, randomised, open-label, multi-centre study, virologically-suppressed PLWH receiving an EFV-containing regimen for ≥12 weeks with ongoing CNS toxicity were switched to DTG and followed-up for 12 weeks (N=40). Plasma neopterin, TRP and KYN concentrations were measured and the KYN/TRP ratio calculated. Rates of CNS toxicities were measured using a questionnaire based on the EFV label and graded according to the ACTG adverse events scale. They included dizziness, depression, insomnia, anxiety, confusion, impaired concentration, headache, somnolence, aggression, and abnormal dreams. Scores ranged from 0 (none) to 3 (severe) and were summed, giving a total score ranging from 0 to 30. CNS toxicity measurements also included assessment using the Instrumental Activities of Daily Living (IADL) and Hospital Anxiety & Depression (HAD) scales. Univariate (paired-samples t-tests) and linear mixed model analyses were conducted. Bonferroni corrections were applied for multiplicity.

The majority of subjects were male (95%) and White (95%). Mean age was 47.8 years. The mean plasma concentration of KYN significantly increased from baseline to Week 12 (2.12 to 2.49 μmol/L, p = 0.002). A non-significant increase was observed for the KYN/TRP ratio (39.7 to 44.8 μmol//mmol, p =0.012). Significant reductions in mean CNS toxicity score (10.1 to 4.5, p < 0.001) and HAD score (14.1 to 8.4, p < 0.001) were observed from baseline to Week 12. Mean IADL scores did not change significantly (7.8 to 7.90, p < 0.570). In the linear mixed model analyses, plasma KYN concentrations and KYN/TRP ratios were found to be statistically significantly negatively correlated with CNS toxicity scores. For every 1 μmol/L increase observed in KYN concentration, a 1.7 point decrease was observed in the CNS toxicity score ( p < 0.019). Likewise, for every 1 μmol/mmol increase observed in the KYN/TRP ratio, a 0.1 point decrease was observed in the CNS toxicity score ( p < 0.027). No significant relationship was observed for KYN or KYN/TRP ratios and HAD scores.

Switching from EFV to DTG was associated with improvements in CNS toxicity and HAD scores, and increases in plasma KYN concentrations. Increases in plasma KYN concentrations and the KYN/TRP ratio correlated with decreases in CNS toxicity. Underlying mechanisms need to be established and may include EFV-induced changes in concentrations of hepatic reactive oxygen species and CNS inflammatory processes.

Immune activation may be associated with reduced immune response to seasonal influenza vaccination in HIV-infected adults

Kitchen M, Kistner O, Wodal W, Gisinger M, Sarcletti M, Zangerle R.

Department of Dermatology and Venerology, Medical University Innsbruck, Innsbruck, Austria, and Baxter Innovations GmbH, Vienna, Austria

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Vaccination against seasonal influenza is recommended for all HIV infected persons, but few data have been reported on seroprotection rates (SP) and geometric mean titers (GMT) in this group. We investigated these parameters in a cohort of 347 HIV infected adults before and 3 months after trivalent seasonal influenza vaccination and studied the effect of age, viral load, CD4 cell count and neopterin level on the immune response. We considered urinary neopterin levels above 250mmol/mol creatinine as marker for elevated immune activation.

69% of patients were male, the median age was 45 years. 297 individuals (85.8%) had a viral load < 50 copies/ml. The median CD4 cell count was 598/μl. Neopterin levels>250mmol/mol creatinine were found in 55 of 347 persons (16,4%) with the highest proportion seen in young individuals up to the age of 30 years (33%). This age group also had the highest proportion of patients with viral loads above 50 copies/ml (52.9%), most of them not yet taking antiretroviral treatment. Elevated neopterin levels were found in 15.8% of patients aged 31–44 years, in 12.5% of 45–59 year-old persons and in 17.6% of patients older than 59 years.

Seroprotection rates for the A/H1N1 and B influenza strains were over 90% in all age groups before vaccination and close to 100% after vaccination. Seroprotection rates for the A/H3N2 strain were lowest in individuals under the age of 30 years both before and after vaccination (23.5% and 50.0%) when compared to older age groups (52.9% and 85.3% in people over 60 years old). Individuals with normal levels of neopterin (minimal immune activation) were more likely to achieve seroprotection against A/H3N2 than patients with elevated immune activation (RR 1.54 (0.74–3.21). The same trend was observed for patients with suppressed viral load compared to those with viraemia (RR 2.32 (0.97–5.56). However, these trends did not reach statistical significance, as the overall number of patients with viraemia and/or high levels of neopterin were low (49 and 55 individuals, respectively).

Changes of neurotransmitters in rat model for tauopathy: role of neuroinflammation

Kovac A, Forgacsova A, Galba J, Majerova P, Novak M (kovac@axon-neuroscience.eu)

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

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Neurotransmitters (NT) are endogenous metabolites that allow the signal transmission across neuronal synapses. Small-molecule neurotransmitters like γ-amino butyric acid (GABA) and glutamate (Glu), and acetylcholine (ACh) and others are critical chemical messengers that transmit signals between neurons and glia. Their biological role is crucial for many physiological functions and their levels are changed in several diseases including the neuroinflammation. The recent studies indicate that neuroinflammation may begin prior to neurodegenerative changes such asloss of synaptic plasticity or degeneration of neurons. To investigate the role of NT in tau induced neuroinflammamtion we developed simple UHPLC/MS method for the simultaneous determination of nine analytes (asparagine, aspartic acid, acetylcholine, GABA, glutamic acid, glutamine, choline, N-acetylaspartate and pyroglutamic acid) in brain tissue. Furthermore we analyzed important metabolites of tryptophan – kynurenine and 3-OH kynurenine, that are both active in CNS. Rat brain tissue was weighed, homogenised with cell crusher in liquid nitrogen, extracted and analyzed without need of derivatization. Because of their high polarity, neurotransmitters were separated on HILIC column and quantified by tandem mass spectrometry. We found signifficant increase/decrease of GABA, pyroglutamic acid, choline, aspartic acid and N-acetylaspartate in brain parts of transgenic animals that are affected by tauopathy process. All these changes can be clearly connected to neuroinflammation.

Depression, quality of life and immune-mediated changes of tryptophan and phenylalanine metabolism in patients with solid tumors

Kurz K, Kink P, Klaunzner M, Willenbacher W, Kasseroler E,4 Weiss G, Holzner B, Fuchs D

Department of Internal Medicine II; Division of Biological Chemistry and of Medical Biochemistry, Biocenter; Psychooncologic Outpatient Department; and Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Austria

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Depression is frequently encountered in patients with cancer, and many patients report about an impaired quality of life. Cellular immune activation directed against the tumor might be involved in the development of these phenomena, as it induces disturbances of tryptophan and phenylalanine metabolism. In our study we wanted to investigate the relationship between immune-mediated changes of amino acid metabolism and changes of mood and quality of life in patients suffering from solid tumors.

Hundred cancer patients (patients with infection or prediagnosed depression were excluded) were asked to answer the EORTC QLQ C30 questionnaire and Beck depression inventory to estimate their quality of life and mood. Serum concentrations of tryptophan, kynurenine, phenylalanine and tyrosine were determined by HPLC, concentrations of neopterin by ELISA, and CRP, liver and renal parameters and blood counts were determined by automated tests. The kynurenine to tryptophan ratios (Kyn/Trp) and phenylalanine to tyrosine (Phe/Tyr) ratios were calculated.

Immune-mediated tryptophan degradation coincided with phenylalanine accumulation. A high proportion of patients (43%) had depressive symptoms (mostly mild) and complained about an impaired quality of life. Depressive patients reported about more symptoms like fatigue, pain, sleep disturbances or loss of appetite and also described a worse emotional, cognitive, social and physical functionality. Patients with an impaired quality of life presented with higher neopterin and CRP concentrations, but lower tryptophan levels. Patients with lower tryptophan concentrations reported about more fatigue, and there were also associations between a decreased physical and social functionality and an impaired role function and low tryptophan concentrations. Women with an increased Phe/Tyr ratio complained about a worse quality of life and had more diarrhea.

Our results suggest that immune-mediated changes of tryptophan and (to a lower degree) phenylalanine metabolism might play a role in the development of an impaired quality of life in patients with solid tumors.

Fungicidal activity of N-chlorotaurine is significantly enhanced in the presence of organic matter including cystic fibrosis sputum medium

Lackner M, Moser I, Gruber M, Nagl M

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria

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N-chlorotaurine (NCT) is a well-tolerated endogenous antiinfective. Due to recent investigations, it is suited for inhalation, too. As patients with infections or chronic diseases of the bronchopulmonary system produce viscous mucus or exudate, the fungicidal and bactericidal activity of NCT was tested in artificial sputum medium (ASM), whose composition mimics the bronchial mucus of CF patients.

Spores of Aspergillus spp., Candida spp., Scedosporium spp. and vegetative forms of bacteria (Staphylococcus aureus, Escherichia coli) were inoculated to phosphate buffer, ASM, or peptone solution. NCT was added at 37°C, and quantitative killing assays were performed.

Conidia of fungi – similar to hyphae - were killed by 55 mM (1.0%) NCT at pH 7.1 and 37 °C with a log10 reduction in CFU of 1 - 4 after 4 h and of 4 to > 6 after 24 h.

In ASM, NCT at a concentration of 0.3 % - 1.0 % showed a microbicidal effect on all fungal species. The impact of 0.5 % and 1 % NCT was significantly - about 50-fold - stronger in ASM than in buffer solution. Fungi were killed after 15 minutes to the limit of detection. Bacteria were killed within 10 min, which was also faster than in phosphate buffer. Moreover, the same was true in 5% peptone solution, where NCT showed an even stronger killing activity than highly reactive halogen compounds. Measurements by means of iodometric titration in ASM showed oxidizing activity over 30 min (60 min) at a concentration of 0.3 % (1.0 %) NCT, which matches the killing tests.

NCT demonstrates microbicidal activity against fungi and bacteria at concentrations ideal for clinical use. Microbicidal activity of NCT in CF and peptone medium was better than in buffer solution, explainable by formation of monochloramine by transhalogenation, which rapidly penetrates microorganisms. These results together with clinical ones render NCT a very promising compound for topical treatment of infections of sensitive body sites.

Gut microbiome and neuroinflammation: an update

Leblhuber F, Schuetz B, Fuchs D

Division of Biological Chemistry, Biocenter, University of Innsbruck, and Biovis Diagnostik MVZ GmbH, Limburg, Germany

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Growing evidence exists on the relationship between the gastrointestinal microbiota and several immune mediated inflammatory diseases, including disorders of the central nervous system.

The microbiota-gut-brain axis is including the brain, glands, gut, immune cells and gastrointestinal microbiota communicating in a multidirectional manner to maintain homeostasis.

Changes in the gut microbiome lead to a broad spectrum of effects including hypothalamic-pituitary-adrenal axis activation, an altered activity of neurotransmitter systems and immune function, and finally to the development of a number of CNS disorders including dementia.

In this update, we focus on the mechanisms by which commensal gut microbiota can regulate function of the microglia and on the involvement of the age related changes of the gastrointestinal microbiome (dysbiosis) and neuropsychiatric symptoms due to the consecutive neuroinflammation.

Installing FVIII specific tolerance in hemophilia via engagement of the aryl hydrocarbon receptor by tryptophan derivatives

Matino D, Gargaro M, Scalisi G, Turco A, Quintana FJ, Puccetti P, Iorio A, Fallarino F

University of Perugia, Perugia, Italy; Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard MedicalSchool, Boston, MA & McMaster University, Hamilton, ON, Canada

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The development of inhibitors is the most serious complication a patient with hemophilia can experience on treatment with clotting factor concentrates. In a cohort of 100 severe hemophilia A patients, we have recently found that the inhibitor positive status was associated with dysfunctional indoleamine 2,3 dioxygenase-1 (IDO1) [1]. The mechanisms whereby IDO1 promotes regulatory effects include production of tryptophan catabolites, collectively known as kynurenines. Some of those tryptophan derivatives are endogenous in nature and act as activating ligands for the Aryl hydrocarbon receptor (AhR), a transcription factor that belongs in the family of basic helix loop helix transcription factors [2]. Specifically, in dendritic cells (DCs), tryptophan catabolite interactions with AhR promote the transcriptional induction of anti-inflammatory cytokines and the emergence of T regulatory (Treg) cells [3]. Here, we report on the potential of tryptophan related AhR ligands for inhibiting the formation of antiFVIII antibodies in hemophilic (F8 KO) mice, a finding that could be important in developing novel strategies to prevent or eradicate FVIII inhibitors.

Methods:Four different endogenous AhR ligands have been selected and used in these experiments. All molecules were administered in vivo to F8 deficient mice at incremental doses and by different routes (orally or intravenously), either concomitantly or after rhFVIII immunization. In specific experiments, AhR knockout mice and co-administration of AhR antagonist were used to analyze the impact of AhR deficiency. To codeliver AhR ligands and FVIII antigens to APCs in vivo and induce antigen specific Tregs, we administered molecules via a nanostructured delivery system. Specifically, we engineered four types of nanoparticles (NPs), 60 nm in diameter, that were stabilized by a polyethyleneglycol (PEG) layer. Overall, we used 1) unloaded NPs, 2) NPs loaded with AhR ligands, 3) NPs loaded with rhFVIII, and 4) NPs loaded with AhR ligands and rhFVIII.

Administration of one of the four AhR ligands prevented the generation of antiFVIII antibodies in nearly 80% of F8KO mice. Similar effects were obtained with both the NPs based approach and the oral delivery of ligands. Of note, the protective effect was negated by coadministration of the AhR antagonist CH223191.

Conclusions:Our results suggest that AhR is a possible molecular partner whereby tryptophan catabolites will control the immune response to FVIII. These findings might lead to novel interventions for preventing or eradicating inhibitors in hemophilia A patients.

[1] Matino D., et al., J Clin Invest 2015;125(10):376681.

[2] Yeste A, et al. Proc. Natl Acad. Sci. USA 2012;109(28):11270–11275.

[3] Bessede A, et al and Fallarino F. Nature. 2014; 511(7508):184– 190.

Association of peripheral blood cell count-derived ratios, biomarkers of inflammatory response and tumor growth with outcome in previously treated metastatic colorectal carcinoma patients receiving cetuximab

Melichar B, Hrůzová K, Kujovská Krčmová L, Javorská L, Pešková E, Solichová D, Hyšpler R, Vošmik M, Bartoušková M, Študentová H

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

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The aim of the present study was to investigate the association of peripheral-blood cell count (PBC)-derived ratios, other biomarkers of inflammatory and biomarkers of tumor growth with the outcome in a cohort of patients presenting for the next line of therapy after the failure of prior lines of treatment. The data of 51 patients with advanced/metastatic colorectal carcinoma treated with cetuximab in the second or higher line of therapy were retrospectively analyzed. Median duration of cetuximab therapy and median survival were 5.1 and 12.1 months, respectively. C-reactive protein (CRP), but not urinary neopterin correlated significantly with PBC-derived ratios. Both CRP and urinary neopterin correlated positively with carcinoembryonic antigen concentrations and biomarkers of liver dysfunction, including bilirubin concentration or AST, ALP and GGT activities. Although a number of parameters predicted overall survival in univariate analysis, only hemoglobin, CEA change and serum bilirubin were independent predictors of survival. In conclusion, in patients with metastatic colorectal carcinoma and predominantly liver metastases, urinary neopterin does not correlate with PBC-derived ratios, in contrast to CRP, but both urinary neopterin and serum CRP concentrations correlate with laboratory parameters of liver dysfunction. The outcome of therapy in the advanced line setting was associated with a decrease of CEA concentration and initial presence of liver dysfunction.

Naturally occurring phyllobilins from plants and their effects on cells: are they overlooked components of human nutrition?

Moser S, Fuchs D, Kräutler B, Gostner JM

Institute of Organic Chemistry, University of Innsbruck, Austria and Division of Medical Biochemistry, Medical University of Innsbruck, Austria

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Chlorophyll breakdown in higher plants proceeds via a common and strictly controlled pathway, resulting in tetrapyrrolic chlorophyll catabolites called phyllobilins. Phyllobilins are ubiquitous in nature; albeit their high abundance, however, there is a substantial lack of knowledge about their physiological properties. Phyllobilins are part of human nutrition and are suspected to have antioxidant properties. We used three different naturally occurring chlorophyll catabolites an investigated their antioxidative properties in both, a chemical test system and in the cell-based antioxidant activity (CAA) assay, using the human intestinal Caco-2 cell line as model system. Our results confirmed antioxidant activities in vitro. In the cell-based system, the yellow chlorophyll catabolite (YCC) in particular showed outstanding bioactivity, e.g. by dose-dependently suppressing the peroxyl radical-induced decay of fluorescein. Importantly, the CAA assay also accounts for the bioavailability of a compound.

When investigating cellular morphology over a broader concentration range, it turned out that concentrations as low as 3 -5 μM were already able to induce morphological changes after 24 h of incubation. The cell’s ability to reduce the indicator dye resazurin, a measure of metabolic activity, was not decreased upon YCC treatment up to a concentration of 45μM.

Further investigations are ongoing using differentiated Caco-2 cells, which allow a better mimicking of the absorptive and defensive properties of the intestinal barrier. We aim to investigate signaling changes on the gene expression level as well as YCC flux using the barrier model.

Biomarkers in transplantation: how specific are they?

Mueller TF

University Spital Zuerich, Department of Nephrology, Zuerich, Switzerland

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Organ transplantation has achieved a remarkable success in early graft and patient survival rates. However, long-term graft function is still not satisfactory and the rate of graft loss over the years has not changed significantly. In particular, chronic rejection starting with a subclinical immune response years before a full manifestation with organ deterioration remains to be a major diagnostic and therapeutic challenge. Hence, biomarkers to detect at an early time point the onset of a significant immune response are needed. In addition these biomarkers should differentiate other causes of graft and patient deterioration, in particular infections and over-immunosuppression to allow for an individualized patient management.

Specific immune monitoring with the detection of HLA-antibodies, measurements of renal function, classification of graft histologies and pharmacodynamic blood level monitoring are examples of personalized medicine already achieved in organ transplantation. Still needed are non-invasive diagnostic markers for subclinical injury, prognostic and predictive biomarkers for graft quality and outcome. Promising data regarding molecular markers of risk for graft failure both in blood, urine and tissue are emerging and examples will be given.

The effects of neopterin on the proliferation and motility of different HCC cell lines

Najjar M, Subashi Y, Valizadeh P, Pournaiem S, Sanaat S, Sahin G, Kunter I

Faculty of Pharmacy, Eastern Mediterranean University, North Cyprus, Famagusta, Turkey

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Increased neopterin levels reflect the activation of the cellular immune system which is of importance in the pathogenesis and progression of various diseases such as carcinomas which include Hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer deaths worldwide. Some studies showed that the concentrations of neopterin are very closely linked with the tumor size in patients with HCC. Generally, cancer patients have high cellular immune activity which can result in increased neopterin levels. However, the effects of neopterin on the biological activity of HCC cells are not yet illuminated. In this study our aim was to understand the effects of neopterin on specific biological activities such as the proliferation and motility of five different HCC cell lines. MTT and SRB assays were carried out to assess the effect of neopterin on the proliferation and wound healing assays were used to evaluate the two-dimensional motility of the cells.

The experimental results showed that the HCC cell lines can be grouped into two categories according to their proliferative response to neopterin, sensetive and resistant. HuH-7, PLC/PRF/5, Hep-3B, SNU449 cells were resistant even under a treatment with high concentrations of neopterin (up to 500μM). On the other hand, SK-Hep1 was the only sensitive cell line among the five different lines used. Even at low concentrations of neopterin, SK-Hep1 showed a statistically significant decrease in the proliferation. Our data also showed a significant increase in the motility of HuH-7, SK-Hep1, and SNU449 cells under the treatment of neopterin but not in PLC/PRF/5 and Hep-3B cells.

This data provides us with variable results concerning the proliferation and motility of the cell lines under different concentrations of neopterin. The underlying molecular mechanisms will be examined in further studies, mainly the effect of neopterin on the signaling pathways and its relation with other biological processes.

Therefore, neopterin might have an important role in the carcinogenesis of HCC and can be a good candidate for the development of new strategies for the treatment.

Alterations of right heart function and iron metabolism in patients with pneumonia

Nicosia F1 Bellmann-Weiler R, Weiss G, Köhler-Lorsbach A, Mair S, Kurz K

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

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Community acquired pneumonia (CAP) is one of the most common infections and can trigger acute cardiac events and be fatal, particularly for the elderly. In our study we wanted to investigate the frequency of right heart dysfunction and a possible correlation with iron metabolism and immune activation in patients with CAP or severe bronchitis.

53 patients (22 women, 31 men, mean age: 65) with CAP or severe bronchitis and echocardiography during their inpatient stay were included in our study. The frequency of right heart dysfunction was high and ranged from 18.2% in patients with no pre-existing cardiac disease (22 patients, 41.5%) to 80% in patients with pre-existing cardiac disorders (5 patients, 9.4%). In the other patient groups with COPD or asthma (11 patients, 20.8%), with severe bronchitis (5 patients, 9.4%) and with the first manifestation of a cardiac complication during CAP (10 patients, 18.9%) the frequency of a right heart dysfunction was respectively 18.2%, 20% and 40%. We observed also a high incidence of electrocardiogram-alterations in our patients with 7 new manifestations of an atrial fibrillation during pneumonia. Patients with pre-existing cardiac disease or with a first manifestation of cardiologic disease during CAP were older and had higher NT-ProBNP levels (both p < 0.05). Patients with right heart dysfunction tended to have higher NT-ProBNP values (p=0.077) and be older (p=0.094). We could also find a correlation between the N-terminal pro brain natriuretic peptide (NT-proBNP) and the duration of in-patient stay (rs=0.380, p=0.008). No significant correlations between right heart dysfunction and iron metabolism as well as immune activation were found.

These findings demonstrate a high frequency of right heart dysfunction in patients with CAP, especially in the elderly and in patients with pre-existing cardiac disorders. Determination of NT-proBNP might be useful in every patient with CAP to estimate the risk for cardiovascular complications or a worse outcome. In patients with elevated NT-Pro BNP levels an echocardiography should be performed- in order to identify and treat patients with cardiac complications.

Ketamine for treatment resistant depression

Nijjar G, McInnes L, Postolache TT

Mood and Anxiety Program, University of Maryland, Baltimore, MD, Kaiser Permanente, Santa Rosa, Kaiser Permanente, San Francisco

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Despite significant progress, depression remains a common and disabling condition that affects millions. Lifetime prevalence of depression is estimated to be around 16% worldwide. Despite a number of alternative effective pharmaceutical agents to treat depression, treatment resistant depression remains a major concern, taking a toll on patients’ quality of life as well as presenting a substantial added societal monetary cost. Over 800 000 people by suicide every year, and a much higher number of individuals with depression report suicidal ideation, that does not satisfactorily respond to commonly used antidepressant agents. Ketamine administration has demonstrated to rapidly and persistently reduce depressive symptoms and suicidal ideation, Low grade inflammation and neuroinfection (such as with Toxoplasma gondii) are known to activate the kynurenine metabolic pathway- leading to the production of quinolinic acid, a neurotoxic NMDA agonist implicated in severe depression and suicidal behavior. Ketamine, in addition to other actions on glutamate receptors, is and NMDA receptor antagonist, and thus, potentially particularly indicated in inflammation mediated depression with suicidality or suicidal behavior. Our presentation focuses on current clinical applications of ketamine, and future studies on prediction of response by molecular markers of inflammation and of the kynurenine pathway.

Serum neopterin concentrations and tryptophan breakdown rates correlate with circulating tumor cells in patients with ovarian carcinoma

Obermayr E, Gostner JM, Fuchs D, Zeillinger R

Department of Gynecology and Obstetrics, Molecular Oncology Group, Medical University of Vienna, Vienna, Austria; Divisions of Medical Biochemistry and Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

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Circulating tumor cells (CTC) are involved in metastasis and are associated with a more severe disease condition in many types of cancer. Several studies discussed the prognostic value of the CTC count in ovarian cancer, highlighting the importance of phenotypic characterization and isolation strategy. In order to further evaluate the information content of CTCs as biomarker in ovarian cancer, we correlated CTC numbers with concentrations of established markers, which have already been shown to increase throughout the stages of progressive malignant diseases, the pterin neopterin and the tryptophan to kynurenine ratio (Kyn/Trp), an estimate of indoleamine 2,3-dioxygenase activity.

Kyn/Trp and neopterin concentrations were increased in those patients with peritoneal carcinomatosis and higher FIGO stage, as well as with the presence of peptidylprolyl isomerase C (PPIC) positive CTCs at baseline. Six months after completion of first-line chemotherapy, neopterin and Kyn/Trp concentrations correlated with CTC counts in patients who had been classified as responders, but not in the non-responder group.

Data suggests that the monitoring of neopterin formation and tryptophan breakdown in combination with CTC counts in cancer patients can be used to monitor disease progression and treatment efficacy. The functional link between peripheral immune activation and presence of CTCs has to be investigated in more detail in order to further optimize anticancer immunotherapeutic approaches.

Tryptophan degradation in pediatric autoimmune hepatitis

Osiecki M, Woynarowski M, Woźniak M, Cukrowska B, Wierzbicka A, Goliszek M Socha P, Janczyk W, Dayanakli D, Fuchs D, Kramp S, Fechner K, Scheper T, Mahler M, Bentow C, Abendroth D, Lytton SD

Warsaw, Poland; Ulm, Lubeck & Munich, Germany; Innsbruck, Austria; San Diego, CA, USA

( )

Kynurenine (Kyn) and the enzymes of tryptophan (Trp) catabolism are predictive biomarkers of T-cell targeting therapies and Treg cells, but their role in pediatric autoimmune hepatitis (AIH) has not been investigated. In this single center cross-sectional survey of tryptophan degradation in Polish pediatric Wilsons Disease (WD, n=8), AIH type 1 (AIH-1, n=40) and AIH type 2 (AIH-2, n=8) the kyn levels; median 2.4 μM range 1.6-3.4 μM (WD), median 2 μM range 0.32-6.1 μM (AIH-1) and median 2.1 μM range 1.4-4.7 μM (AIH-2) and the Kyn/Trp ratios μmole/mmole; median 30 range 20-39 (WD), median 27 range 13-43 (AIH-1) and median 29 range 17-47 (AIH-2) show no significant differences between the patient groups. However, the Kyn/Trp ratios of AIH-1, but not of AIH-2 and WD, negatively correlated with the stage and grade of liver biopsies. Median liver grades of smooth muscle antigen (SMA) autoantibody positive AIH-1; 3 range 0-4 were associated with encephalopathy and significantly higher than that of liver kidney microsome (LKM) autoantibody positive AIH-2; 1 range 0-3 and of autoantibody negative WD; 0.5 range 0-3 (p < 0.01). This study confirms the importance of autoantibodies to distinguish forms of AIH. The low levels of Kyn and the Kyn/Trp ratios within normal range over the period of disease observation indicate minimal or no IFNγ-indoleamine (2,3)-dioxygenase-1 (IDO-1) and/or tryptophan 2,3-dioxygenase (TDO) immune activation. The paradigm of poor IDO/TDO activity leading to Treg cell deficit should be considered as a new paradigm in pediatric AIH.

Biomarker monitoring in AADvac-1 phase 1 clinical trial

Parrak V, Galba J, Forgacsova A, Secnik P, Katina S, Kovac A

Institute of Neuroimmunology, Slovak Academy of Sciences; AXON Neuroscience SE; Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Commenius University, Bratislava; Department of Pharmacology and Toxicology, The University of Veterinary Medicine and Pharmacy, Kosice; SK - LAB spol. s r.o., Lucenec, Slovak Republic; and Institute of Mathematics and Statistics, Faculty of Science, 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 reveals that currently 20 to 30 million individuals suffer from dementia today, with 4.6 million new cases of dementia every year. We did a clinical trial of first-in-man anti-tau vaccine AADvac-1 in patients aged 50-85 years with mild-to-moderate AD at four centers in Austria [1]. A total of 30 patients received AADvac1. Primarily we analyzed titer against immunogen Axon Peptide 108. We achieved a geometric mean IgG antibody titer of 1:31415. Additionally, we monitored several immunological markers including the aromatic amino acids - tryptophan, phenylalanine, tyrosine, kynurenine; thiols – cysteine, homocysteine, cysteinyl-glycine, gluthathione and neopterin. In this paper we discussed the results of longitudinal measurements of these parameters in patients enrolled into AADvac-1 clinical trial. Supported by APVV-14-0547 and VEGA 2/0159/15.

[1] Novak P, et al., Lancet Neurol 2017;16:123-34.

Plasma metabolite changes in chronic lymphocytic leukemia (B-CLL) patients

Parrak V, Galba J, Forgacsova A, Katina S, Melichar B, Fuchs D, Mistrik M, Kovac A

Institute of Neuroimmunology, Slovak Academy of Sciences, Department of Pharmaceutical analysis and nuclear pharmacy, Faculty of Pharmacy of Commenius University, University Hospital Bratislava, and AXON Neuroscience R&D, Bratislava; and Department of Pharmacology and Toxicology, The University of Veterinary Medicine and Pharmacy, Kosice, Slovak Republic; Institute of Mathematics and Statistics, Faculty of Science, Masaryk University, Brno, and Palacký University Medical School and Teaching Hospital, Olomouc, Czech Republic; and Division of Biological Chemistry, Biocenter, Medical University, Innsbruck, Austria

( )

The B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults. The B-CLL is mostly (>75%) diagnosed over the age of 50 years and the majority are man (teenagers occasionally, rare in children). Most people are diagnosed without symptoms as the result of aroutine blood count test with results of high white blood cell count, swollen lymph nodes, spleen, liver, eventually anemia and infections. B-CLL is diagnosed by lymphocytosis, increase of white blood cells, lymphocyte count is greater than 4000 cells per microliter. The diagnosis of B-CLL is based on an abnormal population of B lymphocytes in the blood, bone marrow, or tissues that display characteristic atypical molecular pattern includes the co-expression of cell surface markers CD5 and CD23. In this study we analyzed concentration of 188 metabolites in plasma from B-CLL patients. We found increased levels of amino acids – alanine, glutamic acid and aspartic acid; biogenic amines – spermidine, spermine; phospholipids (C38-C44) and sphingolipids (C20) in plasma a of B-CLL patients. Additionally, glutamine, acylcarnitines (C4, C5, C10), phospholipids (C28-C40) and sphingolipids (C14 and C22) were decreased in plasma of B-CLL patients. We believe that detail understanding of biological pathways behind these changes can lead to development of modern therapeutics for treatment of B-cell chronic lymphocytic leukemia in patients. Supported by APVV-14-0547 and VEGA 2/0159/15.

Plasma vascular endothelial growth factor (VEGF) as potential marker in malignant disease

Petrov J

PZU PAVLINA, private diagnostic laboratory, Skopje, Macedonia

( )

Tumors induce blood vessel growth and this process is known as tumor angiogenesis. The tumor cells secrete various factors, and one which is considered as to have the greatest role is signaling protein vascular endothelial growth factor (VEGF) also known as Vascular Permeability Factor. In various neoplastic diseases it is impossible for tumors to grow and they create metastasis without forming of new blood vessels. So there is a lot of research on blood levels of this signaling protein in many malignant diseases. The aim of this study is to investigate plasma levels in various tumors and to see if it has any possible connection with tumor growth, progression and metastasis.

We collect plasma samples of 40 patients, 16 of them have pancreatic cancer, 16 with gastric cancer, 6 with prostate cancer and 2 with skin cancer. All samples were not frozen and were processed routinely on site. We use Human VEGF Assay kit from Immuno-Biological Laboratories Co. Ltd (www.ibl-Japan.co.jp).

Out of 16 patients with pancreatic cancers, 12 have tumors larger than 3mm3, all 12 of them were in stage III and IV by TNM classification and were very progressive. Ten out of 12 patients have higher levels than 120 pg/mL and the 4 other patients with smaller tumors were negative respectively. In patients with gastric cancer we found higher levels of plasma VEGF in only 3 patients which have positive distant lymph node metastasis. All 6 patients with prostate cancers were postoperative and they did not have metastasis in other organs except one patient have in lymph node. All of them have successful prostatectomy and all have VEGF concentrations below 30 pg/mL. Postoperative patients with melanoma have very high values of plasma VEGF levels (> the 960 pg/ml) but the reason for this was skin transplantation. Generally angiogenesis also begins in transplant acceptance, in contrast patients who have low VEGF levels after transplantation it may indicate incapable transplant acceptance.

In conclusion, blood levels of VEGF in general have large potential as diagnostic but also as prognostic marker.

Intratumoral Th2 predisposition combines with an increased Th1 functional phenotype in clinical response to intravesical BCG in bladder cancer

Pichler R, Gruenbacher G, Culig Z, Brunner A, Fuchs D, Fritz J, Gander H, Rahm A, Thurnher M

Research Group of Urologic Oncology; and Division of Experimental Urology, Department of Urology, and Immunotherapy Research Unit; Division of General Pathology, Department of Pathology; Division of Biological Chemistry, Biocenter; and Department of Medical Statistics, Informatics and Health Economics, Medical University of Innsbruck, Innsbruck, Austria

( )

Intratumoral Th2 predisposition combines with an increased Th1 functional phenotype in clinical response to intravesical BCG in bladder cancer. Th1-type immunity is considered to be required for efficient response to BCG in bladder cancer, although Th2 predisposition of BCG responders has recently been reported. The aim was to evaluate the relationship of Th1 and Th2 components in 23 patients undergoing BCG treatment. Peripheral blood, serum and urine samples were prospectively collected at baseline, during and after BCG. Th1 (neopterin, tryptophan, kynurenine, kynurenine-to-tryptophan ratio (KTR), IL-12, IFN-γ, soluble TNF-R75 and IL-2Rα) and Th2 (IL-4, IL-10) biomarkers as well as CD4 expression in T helper (Th), effector and regulatory T cells were determined. Local immune cell subsets were measured on formalin-fixed, paraffin-embedded cancer tissue by immunohistochemistry to examine expression of transcription factors that control Th1 (T-bet) and Th2-type (GATA3) immunity. We confirmed a Th2 predisposition with a mean GATA3/T-bet ratio of 5.51. BCG responders showed significantly higher levels of urinary (p=0.003) and serum neopterin (p=0.012), kynurenine (p=0.015), KTR (p=0.005), IFN-γ (p=0.005) and IL-12 (p=0.003) during therapy, whereas levels of IL-10 decreased significantly (p < 0.001) compared to non-responders. GATA3/T-bet ratio correlated positively with serum neopterin (p=0.008), IFN-γ (p=0.013) and KTR (p=0.018) after the first BCG instillation. We observed a significant increase in CD4 expression in the Th cell population (p < 0.05), with only a modest tendency toward higher frequency in responders compared to non-responders (p=0.303) [1]. The combined assessment of GATA3/T-bet ratio, neopterin and KTR may be a useful biomarker in predicting BCG response. Th2-promoting factors such as GATA3 may trigger Th1-type immune responses and thus contribute to the BCG success.

[1] Pichler R, et al. Cancer Immunol Immunother 2016 (in press)

Inflammation and suicidal behavior: defining clinical subgroups for meaningful interventional studies

Postolache TT, Brundin L, Groer MW, Peng X, Lowry CA, Fuchs D, Brenner LA

Mood and Anxiety Program, University of Maryland School of Medicine, Baltimore, Maryland, USA; Department of Integrative Physiology and Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado, USA; Military and Veteran Microbiome Consortium for Research and Education (MVM-CoRE); Van Andel Research Institute, Grand Rapids, Michigan, USA; Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria; Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Denver, CO, and VISN 19 MIRECC, Baltimore, USA

( )

Suicidal behavior, a major public health priority, has been increasingly associated with inflammation and conditions leading to elevation in mediators of inflammation, such as infection, autoimmune conditions, allergic disease, brain trauma, endocrine and metabolic factors, and acute and chronic stress. Yet there are conceptual and practical challenges to the hypothesis of inflammation having a mediating role and, thus, being targeted for breakthrough treatments for suicidal behavior. Particularly problematic, a much higher proportion of individuals with positive markers of inflammation and latent infections, such as with Toxoplasma gondii (T. gondii ) than the proportion of individuals who have history of suicidal behavior. Generally, not refining subgroups included for interventional research may lead to falsely negative inflammation-targeting clinical trials, as individuals who are not in need for a corrective intervention are included in the trial. Thus for vertically advancing knowledge in suicide neuroimmunology we have to identify subgroups of individuals at risk for suicidal behavior based on actual documentation of increased inflammation, intersected with clinical (syndromes, sub-syndromes, personality traits), molecular, cellular, neuropsychological, and imaging phenotypes, to specifically design interventions for those subgroups. Such examples based on recent publications and ongoing projects include high CRP blood levels and depression scores, T. gondii IgG and kynurenine blood level, genetic fvariation determining certain enzyme activities on the kynurenine pathway directing the pathway towards neurotoxicity vs. neuroprotection, measures of personality traits of aggression and impulsivity, and neuropsychological tests such as of impaired executive function and/or decision making. For instance- we have very recently reported that low tyrosine levels intersect with T. gondii IgG and personality traits of aggression and impulsivity, and thus tyrosine supplementation could be considered for interventional projects to reduce risk in T. gondii positives with low tyrosine and high impulsivity/aggression scores. Future considerations for subgrouping may be given to neuroimaging (e.g., with inflammation related PET tracers, DTI and connectivity), molecular and behavioral responses to experimental stress, intersections between genetic factors of both host and pathogen in chronic latent infections, and finally, natural immune regulatory variation such as resulting from signals originating within the gut microbiome and gut permeability. Of relevance, our recent data suggest that specific serotypes of T. gondii are associated with certain proinflammatory responses expected to contribute to neurotropism, and with potential immune regulatory signals triggered by a less virulent serotype. Thus, after confirmation on larger studies, treatments for reducing reactivation of T. gondii in high-risk patients could focus on the predominantly neurotropic serotypes triggering ample and persistent immune responses. Information provided these interventional designs is expected to outweigh their likely increased costs and recruitment challenges, as these interventions will be more specifically targeted towards subgroups of patients who need them most, based on specific correctable targets.

Supported by Distinguished Investigator Award from the American Foundation for Suicide Prevention (DIG 1-162-12, T. Postolache); P30 DK072488 NIDDK (NORC pilot/developmental grant PI from the NIDDK, NIH, Bethesda, MD; the Joint Institute for Food Safety and Applied Nutrition, the US FDA, through the cooperative agreement FDU.001418 (T. Postolache); and the MHBA-016-15S, Merit Award from VA CSR&D (T Postolache). The views presented belong to the authors, and do not represent the official views of the NIH, VA, and US FDA.

Immune function, adiponectin, and seasonality of mood and behavior in the Old Order Amish

Raheja UK, Daue ML, Ryan KA, Lowry CA, Brenner LA, Mitchell BD, Postolache TT

Mood and Anxiety Program, Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, USA; Child and Adolescent Psychiatry Residency Program, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA, USA; Program for Personalized and Genomic Medicine, Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Integrative Physiology and Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA; Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC) for Suicide Prevention, Veterans Integrated Service Network (VISN) 19, Denver, CO, USA; Departments of Psychiatry, Physical Medicine and Rehabilitation, and Neurology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. Military and Veteran Microbiome Consortium for Research and Education (MVM-CoRE), Denver, CO, USA

( )

While seasonal changes in mood and behavior (seasonality) have been recognized since antiquity, seasonal affective disorder (SAD), characterized by a predominantly atypical depression syndrome in the fall and winter with alleviation in spring and summer, was described relatively recently [1]. Individuals with SAD experience increased appetite, carbohydrate craving, and weight gain [2], which may be mediated by impaired regulation of adipokines such as adiponectin, lower levels of which having been implicated in obesity, a modern epidemic with multiple medical sequelae. In addition to weight regulation, low adiponectin levels has been associated with both nonseasonal depression in humans [3-6] and shorter photoperiods in photoperiodic mammals [7, 8]. Yet there is no previous research on adiponectin and SAD. In the Old Order Amish the adiponectin gene SNP rs2241766 was previously associated with blood adiponectin levels [9]. Thus, we now examined the association between SAD and seasonality based on the Seasonal Pattern Assessment Questionnaire and a) adiponectin and b) rs2241766 SNP in the Old Order Amish of Lancaster County, PA, with adjustment for age, gender, and BMI.

We evaluated the relationships between the Global Seasonality Score (GSS) and SAD (syndromal or subsyndromal) [10] and a) blood levels of adiponectin (measured by radioimmunoassay) in 636 Old Order Amish adults using multivariate linear methods and b) SNP rs2241766 in 863 participants using a a mixed models approach. We adjusted for age and sex and in further models for BMI.

Participants with SAD had significantly lower adiponectin levels (8.76 ± 1.39 μg/mL) than those without SAD (11.93 ± 0.21 μg/ml) (p=0.01), a finding robust to adjustment for BMI. There was no significant association between GSS and adiponectin (p=0.94). Adiponectin levels were inversely associated with seasonal sleep changes after adjustment for age, gender, and BMI (p=0.014) and with seasonal weight changes following adjustment for age and gender (p=0.002), but not for BMI (p=0.29). Following adjustment for age, gender, and BMI, rs2241766 was associated with GSS (β=0.77, SE=0.30, p=0.01).

To our knowledge, this is the first study of the relationship between SAD and adiponectin. Our results confirm that, as with non-seasonal depression, SAD is associated with lower adiponectin levels, even when accounting for BMI, and that genetic factors may play a role in explaining that association. Modulation of inflammation may be one of the pathways explaining this association, as adiponectin has anti-inflammatory properties via down-regulation of the Th1 pathway [11, 12], and similar to non-seasonal depression [13, 14], measures of immune dysregulation have been reported in SAD [15, 16, 17]. Replicating our findings, particularly with longitudinal designs using repeated measurements across seasons, may lead to better understanding and treating the affective, metabolic, and immune dysregulation of SAD.

The authors would like to thank the staff (in particular, Nancy Weitzel LPN, Mary Morrissey RN, and Theresa Roomet RN), and Amish liaisons at the Amish Research Clinic in Lancaster, PA, for their support. The study was funded by the National Institute of Mental Health of the National Institutes of Health under the K18MH093940 (PI Postolache).

[1] Rosenthal NE, et al., Arch Gen Psychiatry 1984;41: 72-80.

[2] Rosenthal NE, et al., Ann N Y Acad Sci 1987;499:216-30.

[3] Leo R, et al., Neurosci Lett 2006;407:211-3.

[4] Cizza G, et al., J Clin Psychiatry, 2010;71: 1079-87.

[5] Lehto SM, et al., Acta Psychiatr Scand 2010;121:209-15.

[6] Diniz BS, et al., J Psychiatr Res 2012;46:1081-5.

[7] Florant GL, et al., J Comp Physiol B 2004;74:633-9.

[8] Weitten M, et al., Horm Behav 2013;64:611-7.

[9] Pollin TI, et al., Diabetes 2005;54:268-74.

[10] Raheja UK, et al., J Affect Disord 2013;147:112-7.

[11] Wolf AM et al., Biochem Biophys Res Commun 2004;23:630-5.

[12] Tilg H & Moschen AR, Nat Rev Immunol 2006;6:772-83.

[13] Dantzer R et al., Nat Rev Neurosci 2008;9:46-56.

[14] Dantzer R, et al., Psychoneuroendocrinology 2011;36:426-36.

[15] Lam RW, et al., Med Hypotheses 2004;63:567-73.

[16] Leu SJ et al., J Affect Disord 2001;63:27-34.

[17] Song C, et al., J Affect Disord 2015;185:90-6.

How to evaluate Michaelis-Menten-type data in an optimum way? A Monte-Carlo study

Reibnegger G, Paar M, Schrabmair W, Mairold M, Öttl K

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

( )

More than 100 years ago, Michaelis and Menten devised their famous model for simple enzyme kinetics describing the variation of substrate (or product) concentration with time. Integration of this differential equation by separation of variables yields an implicit nonlinear equation which cannot be solved for the substrate/product concentration as an explicit function of time by elementary mathematics. Hence, various linearization schemes such as the Lineweaver-Burk transformation have become enormously popular.

However, since two decades also an explicit solution describing analytically the time-dependent substrate (or product) concentration has been presented [1], which makes use of the Lambert W function [2] and appears to be widely unknown among bioscientists.

In order to promulgate this elegant solution and to put it into context with alternative evaluation techniques, we performed a simulation study. For a simple enzyme reaction, we studied the effects of two sources of measurement error on the parameter estimates KM and vmax obtained by globally fitting time-dependent substrate (or product) concentrations to the explicit and the implicit solution of the Michaelis-Menten differential equation as well as by analysing the same data by nonlinear regression of (initial) reaction velocities versus substrate concentrations, and by the popular linearized versions (Lineweaver-Burk, Hanes-Woolf and Eadie-Hofstee approaches).

The simulations demonstrate a dramatically higher robustness against measurement error for globally fitting the data to the explicit solution, compared with all other evaluation techniques. The reason for the excellent performance of the global fit method versus all approximations evaluating initial velocities versus initial substrate concentrations (i.e., methods avoiding integration of the differential equation) is on the one hand the more efficient utilization of experimental information: while in the approximate techniques each experimental data series describing the initial temporal response to a given initial substrate concentration is compressed to just one single data point, a global regression model enables the inclusion of all measured concentration values versus reaction time. In addition and even more important, the global regression model used offers the option to treat the initial substrate concentration as a free variable during determining KM and vmax; this feature explains the observed superiority over fitting the implicit solution to the data as well as over a standard least square analysis using the explicit solution together with fixed initial substrate concentrations.

After more than a century, the time is ripe to abandon compromises for the analysis of Michaelis-Menten type kinetics. The time-honoured approximations, while having immense historical merits, today can be readily replaced by the more satisfying and elegant explicit solution which in combination with global regression yields accurate and precise system parameter estimates even in the presence of considerable experimental error.

[1] Schnell S & Mendoza C. J Theoret Biol 1997;187:207-212.

[2] Lambert JH. Acta Helv 1758;3:128-168.

Serum kynurenine as a prognostic marker for adverse kidney events after contrast media administration

Reichetzeder C, Heunisch F, von Einem G, Alter M, Kraft R, Kellner KH, Dschietzig T, Kretschmer A, Hocher B

Institut für Ernährungswissenschaft, Universität Potsdam, Nuthetal; Center for Cardiovascular Research, Medizinische Klinik für Nephrologie, Campus Benjamin Franklin, Charité - Universitaetsmedizin Berlin, Berlin; Neuroimmun GmbH, Karlsruhe; Immundiagnostik AG, Bensheim; Bayer Pharma AG, Wuppertal, and Institut für Labormedizin, Berlin; Germany; Department of Basic Medicine, Medical College of Hunan Normal University, Changsha, China

( )

Contrast media induced nephropathy (CIN) remains a serious complication of contrast media enhanced procedures like coronary angiography. There is still a lack of established biomarkers that help to identify patients at high risk for long-term complications [1-3]. Kynurenine is a novel biomarker in cardiovascular disease [4] and complications after exposure due to coronary angiography [5]. The aim of the current study was to evaluate plasma kynurenine as a predictive biomarker for long-term complications of contrast media exposure due to coronary angiography. Long term outcome was measured by the combined endpoint "Major adverse kidney events" (MAKE) up to 120 days after CM application.

A total of 245 patients undergoing coronary angiography were analyzed in this prospective cohort study. Blood and urine samples were obtained at baseline, 24h and 48h after CM application to diagnose CIN. Patients were followed for 120 days for adverse clinical events including death, the need for dialysis, a doubling of plasma creatinine and rehospitalization.

Pre-interventional plasma kynurenine was not associated with CIN. Patients who later developed MAKE displayed significantly increased pre-interventional plasma kynurenine levels (p < 0.0001). Employing ROC analysis revealed that pre-interventional kynurenine is highly predictive for MAKE (AUC=0.838; p<0.0001). The optimal cutoff was found at ≤3.5 μmol/L. Using this cutoff, the Kaplan-Meier estimator demonstrated that concentrations of plasma kynurenine=3.5 μmol/L were significantly associated with a higher prevalence of MAKE until follow up (Chisquare=31.59; <0.0001) This association remained significant in multivariate Cox regression models adjusted for relevant factors of long term renal outcome.

Results of this study showed that pre-interventional kynurenine might serve as a highly predictive biomarker for MAKE up to 120 days after coronary angiography.

[1] Silver SA, et al. Brit Med J 2015;351, h4395.

[2] Stacul F, et al. Eur Radiol 2011;21:2527-41.

[3] Golshahi, J, et al. J Nephropathol 2014;3:51–6.

[4] Polyzos KA & Ketelhuth DFJ. Haemostaseologie. 2015;35:128–36.

[5] Pedersen ER, et al. Eur Heart J 2013;34:2689–96.

Effects of psychiatric rehabilitation on the tryptophan-kynurenine axis

Reininghaus EZ, Reininghaus B, Dalkner N, Riedrich K, Fuchs D

Department of Psychiatry and Psychotherapeutic Medicine, Medical University of Graz, Graz, Austria; TZ-Justus Park Bad Hall, Bad Hall, Austria; Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

( )

The evidence for an association between Tryptophan (Trp) -Kynurenine (Kyn) pathways and neuropsychiatric disorders has increased in the last decades. Nevertheless, there is no clear clinical evidence about changes in these pathways dependent on the current affective status in general psychiatric cohorts. The aim of the study was to measure peripheral changes in Trp, Kyn and Kyn/Trp at the time of admission as well as discharge in individuals undergoing a six-week psychiatric rehabilitation program. 598 individuals with a life-time history of depressive disorders were included, subgroup analyses of euthymic and severely depressed individuals were calculated. Overall, the 6-week psychiatric rehabilitation showed high beneficial effects on the occurrence and severity of psychological and psychiatric symptoms. In line with this, in the whole cohort, Kyn/Trp increased significantly over time in males (0.0284 μmol/L SD 0.006 to 0.0292 μmol/L SD 0.006, p = 0.006). Interestingly, we found no significant changes in female individuals.

In a next step we compared the changes over time dependent on the affective status at the time of admission (euthymic compared to severely depressed individuals). At the time of admission, Trp and Kyn concentrations were significantly lower in depressive compared to euthymic men, while there was no difference at the time of discharge. In males we found a significant group*time interaction for Trp as well as Kyn between euthymic and depressed individuals. In both, the levels decreased in the euthymic group, while they increased in the depressive group over time. There were no significant changes in women.

The results give evidence that a bettering in psychological/psychiatric symptoms goes in line with changes in the Trp and Kyn concentrations also in general psychiatric cohorts. Even in primarily euthymic individuals, with a life time diagnosis of depression, changes could be found over the 6-week period of psychiatric rehabilitation. Gender effects might influence results in general cohorts and should be considered in future analyses.

Alkylglycerol monooxygenase: impact on 3T3-L1 adipogenesis

Sailer S, Keller MA, Hermetter A, Geley S, Golderer G, Werner-Felmayer G, Zwerschke W, Werner ER, Watschinger K

Division of Biological Chemistry and Division of Molecular Pathophysiology, Medical University of Innsbruck, Austria; Institute of Biochemistry, Graz University of Technology, Austria; and Research Institute for Biomedical Aging Research, University of Innsbruck, Austria

( )

Ether lipids comprise a special class of lipids which are not as well characterized as their ester counterparts. They have been shown to be essential for brain structuring, male fertility and are accumulated in fat tissues and models of adipocyte differentiation including 3T3-L1 cells [1,2]. Alkylglycerol monooxygenase (AGMO) is the only known enzyme capable of metabolizing ether lipids such as alkylglycerols and lyso-alkylglycero-phospholipids in a tetrahydrobiopterin dependent manner [3]. We could show that AGMO is expressed and active in a variety of tissues and cell lines including adipose tissue and the murine preadipocyte cell line 3T3-L1. However, the precise role of ether lipids and AGMO in adipose tissue is still elusive.

We generated 3T3-L1 pre-adipocyte cells with knocked down AGMO activity and studied the effect on adipocyte differentiation. We looked at gene expression and protein levels of early and late adipogenic key transcription factors such as CCAAT/enhancer-binding protein β and peroxisome proliferator activated receptor γ.

Our results indicate that AGMO manipulation influences 3T3-L1 differentiation to adipocytes.

[1] Liaw L, et al. J Cell Biochem 2016;117:2182-93.

[2] Gorgas K, et al. Biochim Biophys Acta 2006;1763:1511-26.

[3] Watschinger K & Werner ER IUBMB life 2013;65:366-72.

Urinary neopterin in professional climbers

Schauer M, Filzwieser I, Geisler S, Gatterer H, Fuchs D, Burtscher M

Department of Sport Science, Leopold Franzens University, and Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

( )

In recent years, few studies have been carried out on performance-related parameters in sports climbing [1]. There is an urgent need for further research as there are no established standards available that are suitable for sports-specific performance diagnostics in sports climbing. Currently, the supercompensation model [2] is used for training-control. However, this model was developed exclusively for the endurance range and does not comply with conditions in other training areas. Moreover, no suitable biomarkers are currently available. Already twenty years ago, lactate measurements have been carried out, but the information provided by this parameter is insufficient, e.g. when trying to assess differences between training methods. From several studies elevated neopterin levels were shown to arise after physical stress, whereby its formation is induced by cellular immune reaction [3-6]. Therefore neopterin can possibly be used as a biomarker indicating a preceding acute dynamic load. As urinary testing is possible, the monitoring of neopterin concentrations during training and resting periods would be easy to perform.

Here, we present an explorative study assessing changes in neopterin levels in 22 athletes (12 male and 10 female), who conducted their training in at the Climbing Center Tivoli, Innsbruck, Austria in October to November 2016. Baseline measurements were performed in the morning, and further urine specimens were collected immediately after the workout both on training and non-training (rest) days. Neopterin concentrations were expressed as ratio to creatinine [Neo/Crea] to consider the glomerular filtration rate. In addition the climbing-specific recreational ability was assessed by comparing different force training methods (hypertrophy, intramuscular coordination, short, medium and long-term strength) and their effects on physiological adaptability. The ability to recover was documented by means of a questionnaire (subjective sense of recreation).

No correlation of Neo/Crea with training was found over time, neither for the morning nor for the evening measurments. However, though not significant, there is a trend towards lower neopterin concentrations after training at day 11 compared to day 1. There were no gender differences. Two athletes were excluded from the study due to high baseline neopterin levels which indicates an episode of immune activation not related to the training.

This study is limited due to the low number of partipicants and the difficulty to standardize the physical loads and to judge training efficacy.

[1] Watts PB. Eur J Appl Physiol 2004;91:361-72.

[2] Yakovlev NN. Med Sci Sports Exerc 1975;7:237–47.

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

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

[5] Mrakic-Sposta S, et al. PLoS One 2015;10(11):e0141780.

[6] Strasser B, et al. PLoS One 2016;11(4):e0153617.

Altered neopterin levels in sickle cell anemia

Sabuncuoglu S, Girgin G, Yalcinkaya A, Er Öztas YE, Ünal S, Baydar T

Department of Toxicology, Faculty of Pharmacy, and Department of Clinical Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey; and Department of Pediatrics, Faculty of Medicine, Mersin University, Mersin, Turkey

( )

Sickle cell anemia is the most common inherited haemolytic and pro-inflammatory red blood cell disorder in the world. It results from a mutation in the β-globin gene which causes hemoglobin S to polymerize when deoxygenated, forming rigid polymers within erythrocytes. It has been shown in the recent studies that this disease has implicated invariant natural killer T cells as a critical immune profile which potentiates the condition. Neopterin is produced by monocytes and macrophages as a cellular immune response and it is a byproduct of the tetrahydrobiopterin de novo pathway. There are several clinical conditions associated with elevated kynurenine/tryptophan ratio and neopterin concentrations including inflammatory diseases. In the present study, possible immune activation was investigated via determination of neopterin concentrations and tryptophan degradation in 35 children with sickle cell anemia compared to 20 healthy children. Neopterin, biopterin, kynurenine levels and kynurenine/tryptophan ratio were found to be statistically higher in sickle cell anemia patients compared to control group (p < 0.05). Measuring of neopterin, biopterin, kynurenine and tryptophan levels may be used in sickle cell anemia patients in order to estimate the period of the disease. Therefore, neopterin seems to be a differential biomarker in these patients. Further studies are needed to determine the difference of neopterin levels between the stable phase and crisis period of the disease.

New developments in the transfusion field: does neopterin testing of blood donations still make sense?

Schennach H

Central Institute for Blood Transfusion and Immunology, Tirol Kliniken, General Hospital - University Clinics Innsbruck, Austria

( )

During the last decades the incidence of transfusion transmitted infections with regard to the classical viral infections like HIV, Hepatitis C and B has impressively decreased. In developed countries residual risks of less than 1:2500000 for HIV are common. But since 1985 lots of new viruses have emerged, well known are SARS, West Nile Virus, ZIKA-Virus, and many more which also have an impact on the safety of blood transfusions. When neopterin was introduced in Austria as an additional screening marker on blood donations the main focus was to reduce the width of the diagnostic window phase for known and already via ELISA tested pathogens, but also to detect unknown or not yet tested pathogens. For instance, the research groups in Innsbruck could demonstrate that the diagnostic window phases for HIV and Cytomegalovirus infections were reduced significantly by neopterin. In the following years when more sensitive PCR methods were developed this effect became less impressive. But studies on Tyrolean blood donors showed that especially not routinely tested infectious markers were detected by neopterin elevations (Table 1).

Table 1:

Increased frequency of Parvovirus B19- and EBV-infections in blood donors with elevated neopterin.

The concept of detection of unscreened infectious diseases lies also behind the development of so called pathogen reduction techniques. Different compounds like psoralen derivative amotosalen and riboflavin, which intercalate with the DNA and after irradiation with UV-light prohibit the DNA/RNA-replication of most but not all viruses and bacteria are utilized to prepare “sterile” blood components. At the moment this is feasible for fresh frozen plasma and platelet concentrates but not yet for packed red cells. A slight disadvantage of these methods is that they reduce also to a minor degree the amount of the active components. However, these methods have been widely established in Europe.

A new field for the application of neopterin testing may be a “sentinel” function. Learning from the spreading of ZIKA, Chikungunya and West Nile Virus it makes sense to test blood donors with elevated neopterin levels for potentially intruding pathogens with the aim to establish a risk file for infections of a region. Such a surveillance system is discussed at the moment in the Austrian federal state of Tyrol. But as extensively published neopterin testing provides also data of the health status of a person, which can be of “added value” for the donor.

In conclusion, there are other pathogens than those which are tested by specific tests. The unspecific marker neopterin still reduces the risk but detects not all viral infections and bacterial infections. Esp. low viremic, chronic infections are critical. Neopterin testing is expensive, but reduces the infectious risk of transfusion, is also working on red blood cell concentrates and prevents as an umbrella recently emerging pathogens. And it provides added value for the health of the entire (donor) population.

[1] Schennach H, et al. Clin Chem 1994;40: 2104-5

[2] Schennach H, et al. J Infect Dis 2002;186:1494-7

[3] Schennach H, et al. data from 2004 to 2017 (to be published)

Alkylglycerol monooxygenase phenotyping in Dictyostelium discoideum

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

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

( )

Ether lipids are found in a wide variety of organism like bacteria, protozoa, fungi, higher plants and mammals including humans. Although they are not well studied, it was found that some ether lipids have important antibacterial, antifungal and immune-stimulatory bioactivity. One of the most studied ether lipids is the platelet activating factor (PAF) which is a strong inflammatory mediator. Ether lipids can also raise the permeability of the blood brain barrier thereby improving the delivery of drugs. A central enzyme of ether lipid metabolism is the alkylglycerol monooxygenase. In a tetrahydropteridine-dependent reaction, this enzyme cleaves the ether bond of alkylglycerols and lyso-alkylglycerol phospholipids, including the lyso-platelet activating factor.

Although in general only bilaterial animals express the alkylglycerol monooxygenase, a tetrahydropteridine-dependent alkylglycerol monooxygenase activity was found in the slime mold Dictyostelium discoideum. This organism is called a social amoeba due to cooperation of single celled amoebae in the formation of a fruiting body and spores upon starvation. This differentiation process is characterized by the occurrence of only two main cellular species, and has served as a model to unravel cellular signals such as cyclic adenosine monophosphate (cAMP).

The aim of this study is to investigate the role of alkylglycerol (glyceryl ether) monooxygenase in Dictyostelium discoideum. This will be done by establishing a Dictyostelium discoideum cell line in which the alkylglycerol monooxygenase gene is knocked out and then studying the behavior of the mutant social amoebae during their whole life cycle to characterize the phenotype of the alkylglycerol monooxygenase knock-out strain. Moreover, several lipid classes, using different approaches like thin layer chromatography and liquid chromatography/mass spectrometry, will be analyzed and quantified. These techniques taken together will provide us with a powerful tool to examine the physiological relevance of alkylglycerol monooxygenase in the life cycle and development of the model organism Dictyostelium discoideum.

Preliminary evidence on the relation between urinary kynurenin-tryptophan ratios and sleep variables in a healthy young woman

Singer M, Fuchs D, Hannemann J, Ott M, Bliem HR, Schubert C

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

( )

This integrative single-case study investigates the bidirectional cause-effect relations between Kynurenin-Tryptophan ratio (Kyn/Trp) concentrations and subjective sleep in the natural context of a healthy subject’s everyday life. For 63 days, the 27-year-old woman under study collected her entire urine in two daily 12h fractions (from 8 p.m. to 8 a.m. and from 8 a.m. to 8 p.m.). Kyn/Trp levels of night time urine samples (N=63) were determined via high-performance liquid chromatography (HPLC) technique. Every morning the subject provided information on the following sleep variables by means of a prior-night sleep protocol: sleep quality (SQ), sleep recreational value (SRV), total sleep time (TST), total wake time (TWT) and awakenings during sleep period (ADS). Via time series analysis, consisting of ARIMA modeling and cross-correlational analysis, several significant correlations were identified. Specifically, increases in SQ and TST (positively coded sleep variables) were followed by urinary Kyn/Trp concentration decreases after 48–72h (SQ, lag 2: r=-0.281; p<0.05; TST: lag 2: r=-0.272; p<0.05) whereas increases in TWT and ADS (negatively coded sleep variables) were followed by Kyn/Trp concentrations increases after 24–48h (TWT, lag 1: r=0.267; p<0.05; ADS: lag 1: r=-0.255; p<0.05). No systematic effects in the direction of effects from urinary Kyn/Trp to sleep were observed in this investigation. This study demonstrates that, in a healthy woman, subjective sleep factors trigger urinary Kyn/Trp concentration changes after 24-72h. It thereby provides first insight into the complex interaction between sleep factors and tryptophan breakdown in the natural context of “life as it is lived”.

Changes of inflammation markers with occupational lead exposure

Sipahi H, Girgin G, Palabiyik SS, Tutkun E, Yilm ÖH, Baydar T

Department of Toxicology, Faculty of Pharmacy, University of Yeditepe, Istanbul, Turkey; Department of Toxicology, Faculty of Pharmacy, University of Hacettepe, Sihhiye, and Ankara Occupational Diseases Hospital, Keciören, Ankara, Turkey; Department of Toxicology, Faculty of Pharmacy, University of Atatürk, Erzurum, Turkey and Department of Public Health, Faculty of Medicine, Bozok University, Yozgat, Turkey

( )

Occupational lead (Pb) exposure is well known to cause acute and chronic adverse effects ranging from high blood pressure to symptomatic life-threatening intoxication such as heart disease, kidney disease, and cancer. Although continued efforts to reduce Pb exposures both within and outside the workplace, epidemiological studies continue to provide evidence of health effects even at blood lead levels (BLL) below 10μg/dL. This study was aimed to investigate the changes of inflammation markers with chronic Pb exposure by analyzing neopterin levels and kynurenine (Kyn) to tryptophan (Trp) ratio which reflects indolamine 2,3-dioxygenase (IDO) activity and to compare with healthy volunteers’ parameters. According to our results, mean BLL of the 29 workers was 20 ± 10 μg/dL. Urinary neopterin levels, serum Kyn levels and Kyn/Trp of Pb workers (188 ± 52 μmol/mol creatinine, 3 ± 1 μM, and 43 ± 10 μmol/mmol, respectively) were significantly higher than controls (144 ± 35 μmol/mol creatinine, 2 ± 0.3 μM, and 32±8 μmol/mmol, respectively). Pb exposed workers were divided into further three groups according to their BLLs as 10-19 μg/dL (n= 18), 20-29 μg/dL (n= 8) and 30-49 μg/dL (n= 3). Neopterin levels of the workers with BLL of 30-49 μg/dL were significantly higher than those of BLL with 10-29 μg/dL, while Trp levels decreased. Kyn/Trp of workers with BLL of 30-49 μg/dL were elevated significantly compared to the workers with BLL <30 μg/dL. In addition to neopterin, Kyn and Kyn/Trp levels were positively influenced by Pb exposure. As a conclusion, increased level of inflammation markers confirms the adverse effects of occupational Pb exposure and we suggest that routine monitoring BLLs with inflammation markers could help early diagnosis of possible disorders.

Levels in neurotransmitter precursor amino-acids in patients with depressive syndrome with or without somatic comorbidity

Sperner-Unterweger B, Egeter J, Kohl C, Oberguggenberger A, Meraner V, Gamper E, Hubalek M, Blauth M, Fuchs D, Hüfner K

Department of Psychiatry, Psychotherapy and Psychosomatics; Department of Gynecology and Obstetrics; Department for Trauma Surgery; and Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

( )

A possible connection between chronic inflammation and the development of depression has received increasing attention during the last 10 years. Elevated biomarkers of inflammation, including pro-inflammatory cytokines and neopterin, have been found in depressed patients, and administration of inflammatory stimuli has been associated with the development of depressive symptoms. Furthermore data have demonstrated that inflammatory cytokines can interact with pathways known to be involved in the development of depression, such as the monoamine metabolism [1]. On the other hand the question arises if this possible pathomechanism is relevant in all patients with major depression (MDD) or only in a subgroup.

In order to investigate possible influencing factors we studied the tryptophan-kynurenine and the phenylalanine-tyrosine pathways in physically healthy patients with MDD, in breast cancer patients with and without MDD and in healthy individuals (n=154) [2].

Neopterin, tryptophan and phenylalanine metabolites have been analysed by HPLC or ELISA in 46 MDD patients, 80 breast cancer patients of whom 33 women were also suffering from MDD and 28 healthy matched controls. In another sample consisting of 186 patients admitted to the department of trauma surgery the same neurotransmitter pathway –metabolites have been measured. Concomitantly we assessed symptoms of depression and anxiety in these patients.

Significantly higher serum neopterin concentrations were found for patients with MDD (p=0.026) as a marker of Th1-related inflammation. The kynurenine/tryptophan ratio (index of the serotonin pathway) was significantly affected by the factors “breast cancer” and “state anxiety” and their interaction (p<0.001, p=0.026, p=0.02 respectively). The phenylalanine/tyrosine ratio (index of the catecholamine pathway) was affected by the factors breast cancer” and “diagnosis of depression” and their interaction (all p<0.001). The difference in neurotransmitter precursor metabolism was most evident in patients after trauma surgery with a comorbidity of depression reflected by a significantly increased phenylalanine/tyrosine ratio (p=0.008). The kynurenine/tryptophan ratio (index of the serotonin pathway) was not affected in this group. In conclusion, alterations of neurotransmitter precursor monoamines were observed in patients with breast cancer as well as in trauma patients and they seem to influence mental health.

[1] Sperner-Unterweger B, et al. Prog Neuropsychopharmacol Biol Psych 2014;48:268-76.

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

Molecular architecture of pterin deaminase from Saccharomyces cerevisiae 3458

Thandeeswaran M, Karuppuswamy V, Kiran KG, Ayub Nawaz KA, Mahendran R,Palaniswamy M, Angayarkanni J

Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India; Department of Microbiology, Karpagam University, Coimbatore, Tamil Nadu, India

( )

Over a period of 40 years, enzymes are employed as chemotherapeutic drugs for disease and conditions. Enzymes have the specific affinity and binding proportion towards particular targets with minimal toxicity. This makes them an ideal drug for treating different clinical disorders. Cancer cells are more sensitive to enzymes than a normal cell because enzymes dissolve fibrous coating on cancers cells, allowing the immune system to work. Earlier reports have confirmed anticancer activity of pterin deaminase in Fungal, bacterial and Slime mold genera [1]. With this information this attempt was made to divulge the presence of pterin deaminase in Yeast. Yeast has been a pioneering model for studying the regulation of eukaryotic metabolism.The present study was attempted to purify and characterize pterin deaminase enzyme from Saccharomyces cerevisiae. The protein content in the extracellular extract was collected by ethanol precipitation method. Partial purification of pterin deaminase enzyme was achieved by Ion exchange chromatography (Hi-Trap QFF) by Fast Protein Liquid Chromatography (AKTA Purifier) [2]. The molecular weight of the protein was apparently determined by SDS-PAGE and the presence of pterin deaminase was confirmed by activity staining [3]. The purified enzyme was further biochemically characterized. Molecular docking studies showed higher binding affinity towards folic acid interaction [4]. It is confirmed, the amino acid involved in the pterin deaminase is taking -NH3 group to confirming deamination property [5].The structure of this protein may open the windows for new drug targets in cancer therapy.

[1] Kusakabe H, et al. Agric Biol Chem 1979;43:1983-4.

[2] Rembold H, et al. Biochim Biophys Acta1969;184:589-96.

[3] Takikawa S, et al. J Biochem 1979;85:785-90.

[4] Fan H, et al. J Am Chem Soc 2013;135:795-803.

[5] Kakebeeke PI, et al. J Bacteriol 1980;143:307-12.

Amino acid and worldview: phenylalanine, sports, and crisis of meaning

Vötter B, Böhmer M, Georgi M, Spitzenstätter D, Geisler S, Fuchs D, Schnell T

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

( )

A dysfunctional amino acid and subsequent neurotransmitter metabolism is often connected with a decreased quality of life. Anyhow, psychoneuroimmunological processes are not yet clearly understood. Several studies show links between a lack of meaning in life and higher mortality risk, probability to develop cardiovascular and Alzheimer diseases, cortisol release, increased levels of cytokines, C-reactive protein, etc. [1] The aim of the present study was to clarify the association of the biological parameter phenylalanine (a precursor of the neurotransmitter dopamine) with a negative dimension of worldview defined as crisis of meaning. A sample of N=134 students from the University of Innsbruck completed a questionnaire and participated in saliva collection. Crisis of meaning was measured by the appropriate scale from the Sources of Meaning and Meaning in Life Questionnaire (SoMe [2]; Cronbachs-Alpha=0.94). Subsequently, a biochemical analysis of phenylalanine was conducted. The median level of phenylalanine was 62.5 μmol/L, the average deviation 33.1 μmol/L. 85% of the sample were female; the age ranged from 18 – 37 years (mean=19, SD=3.6). All covariates (e.g. sex, age, coffee/nicotine/alcohol/sleep duration before data collection, medication, size, weight, family status, use of hormones, diseases) except for frequency of sports were insignificant in predicting crisis of meaning. A multiple linear regression analysis was used to test if the biochemical parameter phenylalanine and frequency of sports significantly predicted participants’ crisis of meaning. The results of the regression indicated the two predictors explain 12% of the variance (R2=0.12, F (2,128)=8.61, p<0.001). It was found that phenylalanine significantly predicted crisis of meaning (β=-0.16, p<0.03), as did the frequency of sports (β=-0.31, p<0.001). The results highlight that the saliva concentrations of amino acid phenylalanine as well as frequency of sports independently serve as predictors for existential conflicts.

[1] Schnell T. Psychologie des Lebenssinns 2016.

[2] Schnell T. J Pos Psychol 2009;4(6):483-99.

Effects of systemic iron perturbation on the peripheral metabolism and mitochondrial fitness

Volani C, Demetz E, Doerrier C, Gnaiger E, Paglia G, Weiss G

Dept of Internal Medicine II, Medical University of Innsbruck, and Oroboros Instruments, Innsbruck, Austria; an dEURAC, Center for Biomedicine, Bolzano, Italy

( )

Mitochondria are dynamic organelles, involved in different cellular processes, including oxidative phosphorylation. Iron is a fundamental co-factor in these processes, being not only part of the mitochondrial complexes of the electron transport system but also of aconitase, central enzyme of the citric acid cycle. Hence, systemic iron perturbation could affect mitochondrial fitness as well as the overall metabolism. Nevertheless, how iron imbalances affect mitochondrial activity and cellular metabolism is poorly understood. Therefore, we aimed to determine the impact of alterations in iron homeostasis on the mitochondrial function and the peripheral metabolites.

Mitochondrial fitness was studied in human PBMCs. Shortly, human PBMCs were collected from buffy coats, purified cells (2x10^6 cells/ml) were resuspended in mitochondrial respiration medium (MiR05), and mitochondrial respiration was assessed by high resolution respirometry (OROBOROS Instruments, Austria). Peripheral metabolites were isolated from mouse whole blood; metabolomics analysis was performed by using liquid chromatography mass spectrometry (LC-MS).

Our ongoing experiments indicate that mitochondrial function testing can be successfully performed in human PBMCs. We are currently analyzing the mouse peripheral blood metabolites. Lastly, combining the use of high-resolution respirometry with the analysis of the peripheral blood metabolites should provide useful information about changes in the overall metabolism.

About the article

Published Online: 2017-04-20

Published in Print: 2017-05-01


Citation Information: Pteridines, Volume 28, Issue 1, Pages 37–58, ISSN (Online) 2195-4720, ISSN (Print) 0933-4807, DOI: https://doi.org/10.1515/pterid-2017-0006.

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