41^th International Winter-Workshop Clinical, Chemical and Biochemical Aspects of Pteridines and Related Topics Innsbruck, Austria, February 5^th to 7^th, 2025

Oxygen-dependent regulation of NAD and hypoxia conditioning: a review

Johannes Burtscher, Martin Kopp

Department of Sport Science, University of Innsbruck, Innsbruck, Austria; Department of Sport Science, University of Innsbruck, Innsbruck, Austria (Johannes.Burtscher@uibk.ac.at)

Cells of aerobic organisms need to protect themselves against hypoxia (reduced oxygen availability). Sophisticated biochemical and molecular mechanisms are in place to quickly sense and respond to acute hypoxia and to induce cellular adaptations for the management of future hypoxic stress. Nicotinamide adenine dinucleotide (NAD) metabolism is highly sensitive to changes in cellular oxygen levels and mediates biochemical responses to hypoxic stress. It tightly interacts with molecular pathways coordinating adaptations aimed at increasing the cellular resilience to hypoxia, notably the hypoxia-inducible factors (HIF) pathways. The potential of modulating cellular adaptations to hypoxia in order to improve human health or to interfere with pathological processes is increasingly recognized. Pharmacologically targeting HIF signaling or directly applying controlled hypoxic stress (termed hypoxia conditioning) are promising avenues for harnessing the power of hypoxia-induced adaptations in a plethora of diseases, including inflammatory, metabolic and neurodegenerative diseases. Although NAD metabolism is greatly responsive to hypoxia and a powerful regulator of cellular metabolism and redox state, its role in the health benefits conferred by hypoxia conditioning has been scarcely explored. Here we present a literature analysis of the alterations of NAD metabolism following reduced oxygen availability and put these changes in the context of health-promoting hypoxia conditioning.

Studies in animals demonstrate a bidirectional interdependence of NAD metabolism and hypoxia responses. Hypoxia acutely leads to an accumulation of the reduced form of NAD (NADH), an effect that can cause reductive stress but usually is reversible and may support the induction of mechanisms improving cellular resilience to hypoxia. Conversely, severe prolonged hypoxia, especially if interspersed with periods of reoxygenation, reduces overall NAD levels and increases the NADH/NAD+ ratio (NADH hyperoxidation), an effect associated with permanent mitochondrial, cell and tissue damage. Controlled exposure to moderate hypoxia, however, mitigates detrimental reductions in NAD in subsequent severe hypoxia/ischemia, suggesting that protective adaptations to hypoxia include improved control of NAD homeostasis. In addition, a substantial body of literature indicates that preservation of NAD-levels by NAD supplementation is a potent means to protect from hypoxic damage.

Taken together, the interplay of NAD metabolism and other components of the cellular hypoxia-response system (e.g., HIF pathways) appears to be crucial for the management of hypoxic stress and the protective outcomes of hypoxia conditioning. Targeted pharmacological modulation of NAD metabolism (e.g., NAD supplementation) is an emerging possibility to control the hypoxic stress response, while hypoxia conditioning might be a novel non-pharmacological strategy to modulate NAD metabolism.

Social stress and feather pecking behaviour alter tyrosine, phenylalanine, and kynurenine concentrations in the plasma of laying hens

A. Lea Nicklas, Johanna M. Gostner, Lucia Parráková, Cornelia A. Karg, Nienke van Staaveren, A. Michelle Edwards, A. Kate Shoveller, Paul Forsythe, Alexandra Harlander

Department of Animal Biosciences, Campbell Centre for the Study of Animal Welfare, University of Guelph, Guelph, Canada; Institute of Medical Biochemistry, Medical University of Innsbruck, Innsbruck, Austria (anicklas@uoguelph.ca)

During severe feather pecking (FP) behaviour, one hen repeatedly pecks at the feathers of another, which may result in feather damage, loss, or skin injuries. Severe FP is highly prevalent and is associated with an altered gut microbiota and neurotransmission. The gut microbiota can be modified with prebiotic galacto-oligosaccharides (GOS) which may promote bacteria that produce and metabolize tryptophan, tyrosine (Tyr), and phenylalanine (Phe), respectively. These amino acids are important precursors for central serotonin and dopamine production.

This study aimed to investigate how GOS supplementation and social stress affect FP behavior, and whether GOS, social stress, and FP phenotype influence plasma aromatic amino acid and kynurenine (Kyn) concentrations.

A total of 191 White Leghorn hens from genetic lines selected for high (HFP) or low (LFP) FP activity were housed in 12 pens with equal numbers of HFP and LFP birds. From 29 to 31 weeks of age, six pens were offered water supplemented with Nutrabiotic® GOS (74% GOS; Dairy Crest Limited, Davidstow, Cornwall, United Kingdom) while the other six received plain water. Half the pens in each water treatment group experienced social stress through repeated mixing of unfamiliar hens. All pens were video-recorded, and blinded observers analyzed FP behaviour. Birds were assigned a phenotype of extreme, moderate or no FP based on the number of severe FP bouts recorded. At the end of the treatment period, blood plasma samples were collected from all birds. Amino acid concentrations were measured using high-performance liquid chromatography, while enzyme-linked immunosorbent assay was used to determine Kyn levels. Generalized linear mixed models (SAS V9.4) were used to assess the effects of supplementation, stress, and FP phenotype on Tyr, Phe, and Kyn concentrations.

There was no significant effect of GOS supplementation, however, social stress increased Tyr concentrations (p = 0.0374). Hens who exhibited extreme FP had lower Tyr (p = 0.0012) and Phe (p = 0.0190) concentrations and a higher Phe:Tyr ratio (p = 0.0322) than birds who pecked moderately or not at all. A significant interaction between stress and phenotype resulted in lower Kyn (p = 0.0163) concentrations in unstressed hens that did not exhibit FP (LSM 0.22 ± 0.01) compared to stressed hens with moderate (LSM 0.31 ± 0.01) or extreme (LSM 0.26 ± 0.02) FP behaviour.

These findings align with previous studies suggesting that amino acid metabolism is closely linked to social stress and FP behaviour and indicate that extreme feather peckers have distinct neurochemical profiles which may be exacerbated during times of social stress. While GOS did not alter amino acids during stress, follow-up studies will investigate its potential impact on stress recovery.

Multi-method analysis of a natural sickness period: An integrative single case study

Christian Schubert, Lennart Seizer, Nina Camille Sophia Lauby, Dietmar Fuchs

Department of Psychiatry, Psychotherapy, Psychosomatics and Medical Psychology, Medical University Innsbruck, Austria; Institute of Biological Chemistry, Medical University of Innsbruck, Innsbruck Austria (christian.schubert@i-med.ac.at)

Sickness behavior is a coordinated set of adaptive psychological changes mediated by immune system activity, e.g., during infection and wounding. In this integrative single-case study, a 27-year-old healthy woman collected her entire urine over a period of 63 days in continuous 12-h intervals (126 measurements in total). In addition, among other regular psychological assessments, she completed an emotional state questionnaire each morning and evening and had an in-depth psychological interview once a week. In the urine samples, neopterin, cortisol and creatinine were measured. Coincidentally, the patient experienced a period of sickness during the study interval, lasting two days (from 12-h interval 21 to 24), accompanied by fatigue, vomiting, diarrhea, fever, and increases in urinary neopterin and cortisol concentrations. During this sickness period, structural breaks occurred in the time-series of two positive emotional states, i.e. performance-related activity and general feeling of comfort, with significant drops in their mean levels lasting for over 50 days until the end of the study. The extended biopsychosocial study data enable a broad discussion of these results from socio-immunological and bio-psychological perspectives.

Neuroimmune profiling in the EULAST cohort – associations of immunometabolic markers and psychopathology

Celina Wilgermein, Johanna M. Gostner, Stefanie Hofer, Cornelia A. Karg, Fabienne Post, Hubert Hackl, Barbara Sperner-Unterweger, Alex Hofer

Division of Psychiatry I and Division of Psychiatry II, Department of Psychiatry, Psychotherapy, Psychosomatics and Medical Psychology, Medical University of Innsbruck, Innsbruck, Austria; Institute of Medical Biochemistry and Institute of Bioinformatics, Medical University of Innsbruck, Innsbruck, Austria (celina.wilgermein@i-med.ac.at)

This sub-project of the European Long-acting Antipsychotics in Schizophrenia Trial (EULAST), which compared long-acting and oral antipsychotic treatment (aripiprazole/paliperidone) in premedicated individuals diagnosed with schizophrenia by applying a longitudinal, randomized, pragmatic approach, aimed to identify a potential neuroimmune profile by investigating associations between inflammatory parameters, symptomatology, and treatment response.

Serum levels of pro-and anti-inflammatory cytokines, brain-derived-neurotrophic factor (BDNF) and neurotransmitter precursor amino acids and derivatives were measured in samples from a total of 360 schizophrenia patients from different study centers. Symptomatology was assessed using the Positive and Negative Syndrome Scale (PANSS). The impact of inflammatory parameters on psychopathological outcome was analyzed using mixed effect models, linear and logistic regression analyses.

After correction for multiple testing and taking confounding covariates into account, we observed a significant negative effect of kynurenic acid as well as a significant positive effect of interleukins (IL)-2, IL-8, and IL-18 on the PANSS total score. By applying hierarchical cluster analyses based on age, gender, BMI and symptomatology we did not identify distinct subgroups.

In summary, these interim results point to a neuroimmune profile heterogeneity in premedicated schizophrenia patients and could be interpreted as a normalization of inflammatory signs with antipsychotic treatment.

Impact of PFAS on neopterin formation and tryptophan breakdown in human PBMCs

Dorina E. Kernbichler, Lucia Parráková, Cornelia A. Karg, Thomas K. Felder, Anita Siller, Harald Schennach, Maria Uhl, Johanna M. Gostner

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, and Core Facility Metabolomics II, Biocenter, Medical University of Innsbruck, Austria; Institute of Pharmacy, Department of Pharmacognosy, University of Innsbruck, Innsbruck, Austria; Central Institute of Blood Transfusion and Immunology, University Hospital of Innsbruck, Innsbruck, Austria; Department of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria; Environment Agency Austria (Umweltbundesamt GmbH), Vienna, Austria (johanna.gostner@i-med.ac.at)

Per- and polyfluorinated substances (PFAS) are pervasive anthropogenic environmental contaminants. Due to their water-, grease-, and stain-resistant properties, they are widely used across various industries. However, their high thermal and chemical stability render them highly persistent both in the environment and within the human body. In recent years, concerns regarding the toxicity of PFAS have grown, particularly regarding their potential immunotoxic effects.

In this study, human peripheral blood mononuclear cells (PBMCs) from healthy donors, either stimulated or unstimulated with the mitogen phytohemagglutinin, were exposed to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS). The study aimed to assess the potential effects on immunobiochemical pathways involved in cellular immune activation, focusing on neopterin formation and tryptophan breakdown.

While stimulated PBMCs exhibited increased resistance to PFAS treatment, significant alterations in neopterin concentrations and the kynurenine metabolite profile were observed in unstimulated cells.

Evidence from experimental and in vivo studies increasingly supports a multifaceted impact of PFAS on immune regulation. In line with this, data from the present in vitro study suggest that exposure to PFOA and PFOS interferes with immune-regulatory metabolic pathways linked to cellular immune activation.

In vitro characterization of the immunotoxicity of BPA and BPA alternatives using pathway-level dose-response modeling with DoseRider

Pablo Monfort-Lanzas, Lucia Parráková, Oriol Ruiz Catalan, Anita Siller, Harald Schennach, Cornelia A. Karg, Hubert Hackl*, Johanna M. Gostner*

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Austria; Department of Pharmacognosy, University of Innsbruck, Innsbruck, Austria; Central Institute of Blood Transfusion and Immunology, University Hospital of Innsbruck, Innsbruck, Austria; Institute of Bioinformatics, Biocenter, Medical University of Innsbruck, Austria (*equal contribution) (pablo.monfort@i-med.ac.at)

Bisphenol A (BPA) and its analogs are known endocrine disruptors with increasing evidence of immunotoxicity. Previous studies have shown BPA’s suppression of IDO-1 activity and Th1-type immune responses. However, the impact of BPA alternatives on immune regulatory pathways remains insufficiently characterized, particularly regarding dose-response behavior at the pathway level.

We reanalyzed RNA-seq data (MCF-7 cells, GSE211183) exposed to BPA and 15 structural analogs. Four compounds (BPA, BPAF, BPS, 4,4’-BPF) were selected for in-depth analysis. A IDO-1 signature comprising 281 genes was constructed from multiple literature-derived gene sets associated with IDO-1 activity. Pathway-level dose-response modeling was performed using DoseRider, which applies spline-based mixed models to detect non-monotonic trends. Enrichment analysis was used to evaluate tryptophan metabolism, immune response, and cell cycle pathways.

From the differential expression analysis, 200 genes were found dysregulated in all bisphenol compounds. These genes are primarily involved in immune response suppression and cell cycle activation. IDO-1 signature analysis revealed two distinct gene clusters: one upregulated (proliferation-related) and another downregulated (immune regulatory). All four compounds showed dose-dependent effects on tryptophan metabolism, with positive enrichment scores, and consistent downregulation of immune checkpoints. DoseRider modeling shows pronounced non-linear dose-response patterns, with mid-range doses exerting stronger effects than higher doses. BPA and BPAF exhibited the most potent immunotoxic effects.

Our results demonstrate that BPA analogs share immunosuppressive and proliferative transcriptional profiles with BPA, significantly impacting IDO-1-linked immune pathways. Non-monotonic dose-response relationships highlight the inadequacy of linear models in endocrine toxicology. DoseRider provides a robust framework for detecting subtle immunotoxic effects, supporting its utility in regulatory risk assessment of bisphenol compounds.

Investigation of the UV protective potential of secondary metabolites from natural sources in human cornea cells

Cornelia A. Karg, Fabian Hammerle, Johanna Lückenbach, Michael J. Zwerger, Simone Moser, Markus Ganzera, Johanna M. Gostner

Institute of Pharmacy, Department of Pharmacognosy, University of Innsbruck, Innsbruck, Austria; Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Austria (cornelia.karg@uibk.ac.at)

Ultraviolet (UV) radiation is an omnipresent environmental factor with significant health implications, causing damage to skin and ocular tissues. Prolonged UV exposure increases the risk of skin cancer and premature aging, as well as eye diseases like cataracts. While traditional UV protection relies on synthetic chemical agents, interest in natural alternatives is growing due to their efficacy and lower environmental impact.

Mycosporine-like amino acids (MAAs) are small, water-soluble compounds found in organisms like red and green algae, as well as cyanobacteria, and are capable of efficiently filtering UV radiation. They have also been detected in marine invertebrates and fish, likely through dietary transfer. Notably, high concentrations of MAAs have been found in the eyes of fish from the Baltic Sea, suggesting their role in protecting visual function against UV radiation.

In this study, the UV-protective effect of MAAs was investigated in a 2D model of human corneal epithelial cells. Using a certified sunlight simulator, a protocol was developed to assess UV-induced formation of reactive oxygen species (ROS). Known natural and synthetic sunscreen compounds were used as controls. MAAs, such as porphyra-334, significantly reduced UV-induced ROS formation in a dose-dependent manner, without impairing cell viability.

The eyes are highly susceptible to UV-induced oxidative stress, with the human cornea absorbing approximately 90% of UV-B radiation. Although our results are preliminary, they suggest that MAAs may be promising natural protectants for UV-sensitive human tissues, such as the skin and corneal epithelium; however, a further investigation of the underlying mechanisms is required.

Laboratory medicine in palliative care: Is there a role for neopterin and related biomarkers?

Bohuslav Melichar

Department of Oncology, Palacký University Medical School and University Hospital, Olomouc, Czech Republic (bohuslav.melichar@fnol.cz)

Along with the advances of medical and surgical therapies comes, somewhat paradoxically, a major increase in the number of patients with incurable disorders that can be explained by aging of the population and prolongation of survival in chronic disorders. Historically, most medical therapies have been of palliative nature well into the 20th century. The advent of causal medication has changed the landscape, first in infectious diseases and later in cancer. Effective treatments have emerged for many chronic disorders, including diabetes mellitus, coronary heart disease or chronic obstructive lung disease. These advances lead to a tendency for denial of mortality as an integral part of human existence. Death was perceived as a failure of the treatment strategy or the medical team. Palliative medicine emerged as a response to failure of a purely technological approach to address the needs of humans with terminal illness. The essence of palliative medicine is adjusting the interventions that include not only medical therapy, but also psychological, social or spiritual support to the changing goals of care during the course of disease.

Laboratory methods are important in assessing the condition of patients across a spectrum of different disorders and throughout the course of the disease from early diagnosis to the terminal phase. In a holistic concept of patient care, laboratory values that reflect the organ function are essential in the assessment of the current status as well as the prognosis. Practically all of the disorders in the palliative care practice are associated with the activation of the systemic immune and inflammatory response. Thus, the determination of the biomarkers of the activation of inflammation and immune response may be instrumental in the assessment of current status, disease dynamics and prognosis.

The determination of neopterin in this setting may be of advantage because of the possibility to utilize sample matrices such as urine that can be sampled in a non-invasive manner repeatedly. However, the potential of laboratory biomarkers of immune system activation in the management of patients in the palliative care setting is still not fully recognized. Neopterin concentrations in urine, serum or other biological fluids are increased in different chronic disorders. While this lack of specificity may be viewed as a disadvantage in the diagnostic phase, in patients with advanced disease and often multiple comorbidities the determination of neopterin may facilitate an assessment of general condition of the patient resulting from different disorders. Prognostic information as well as understanding of individual disease dynamics may offer an invaluable aid in guiding the patient management.

More studies should address the potential of neopterin or related biomarkers, such as kynurenine and tryptophan, in daily practice of palliative medicine, including the patients in the terminal phase of the disease.

A preliminary study on neopterin, tryptophan and kynurenine levels of human colostrum samples

Gözde Girgin, Sinem Meric Deliveli, Sonia Sanajou, Terken Baydar

Department of Toxicology, Faculty of Pharmacy, Hacettepe University, Lokman Hekim University, Ankara, Turkey (gzdgirgin@gmail.com)

Colostrum is a biological fluid produced by humans, cows, and other mammals before breast milk is released. It is very nutritious, contains high levels of antibodies and promotes growth, obtains immunity and improves gut health in newborns. A number of studies evaluated the neopterin status in various human biological samples and tryptophan and kynurenine levels in blood, but rarely in human milk.

The aim of the present study was to detect neopterin concentrations and the ratio of kynurenine to tryptophan in human colostrum milk samples.

Neopterin levels of the milk samples were determined by the commercial enzyme linked immunoassay while kynurenine and tryptophan levels were analysed by high performance liquid chromatography method. Samples were collected in the first breastfeeding time as seventeen colostrum samples (Tekirdag Star Medica Hospital, Ethics Committee approval number #08.29.2019) and stored at -20°C until analysis.

It has been determined that the mean neopterin levels and the kynurenine to tryptophan ratios in colostrum samples were 4.7- and 4.4-fold higher than the childhood reference serum levels, respectively. No significant relationship was found among the measured parameters in the samples (all, p>0.05).

The limitation of this study is that the serum parameters were not evaluated as blood samples were not collected from new mothers.

A validated HPLC-MS/MS method for the quantification of melatonin in human breast milk samples

Kevin Allmer, Johanna Pichler, Emma Lanzinger, Silke Häusler, Thomas Felder

Department of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria (t.felder@salk.at)

Melatonin is an endogenous neurohormone generated from L-tryptophan through a sequence of enzymatic reactions prior its secretion. During the prenatal phase, the human fetus lacks endogenous melatonin production and relies on maternal melatonin release through transplacental transfer. Moreover, neonates do not produce endogenous melatonin in the initial months of life and consequently depend on exogenous sources from maternal breast milk. The diurnal variations in melatonin levels in breast milk facilitate the establishment of circadian rhythms in the neonate, which appears essential for brain development. Moreover, premature infants demonstrate a more pronounced impairment of melatonin physiology, despite greater dependence on the antioxidant qualities of melatonin.

We established and validated a highly sensitive LC-MS/MS method employing liquid-liquid extraction with ethyl acetate to detect endogenous melatonin concentrations in breast milk at low pg/ml levels. Method validation was conducted in accordance with the Eurachem Guideline “The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics (2nd ed. 2014).” The extracts were analyzed using an Exion LC linked to a Triple Quad 5500+ mass spectrometer (Sciex). The validated approach was utilized on breast milk samples from mothers of pre-term newborns, collected during nighttime (n=40) or daytime (n=40). The limit of detection (LOD) for melatonin was 1.4 pg/ml, while the lower limit of quantification (LLOQ) was 4.8 pg/ml, covering the range observable in maternal milk samples. Daytime and nighttime samples exhibited differences, with melatonin levels in nighttime samples being significantly elevated to daytime levels (p<0.0001; mean-day: 8.9 pg/ml; SD: 16.8; mean-night: 27.5 pg/ml; SD: 10.0). The parameters selectivity, linearity, inter- and intra-day precision, accuracy, and ion suppression effects were considered acceptable in accordance with the validation guidelines.

The determined melatonin concentrations in breast milk samples were consistent with previously published values. We also verified elevated amounts in breast milk during nighttime samples. There is a deficiency of longitudinal evidence about the variations in melatonin secretion during postnatal follow-up in preterm human milk, relative to gestational age and mother pregnancy-related illness. The established method can be applied to longitudinally examine (rhythmic) melatonin release in breast milk after preterm birth. Delivering optimal care to preterm neonates, while understanding the importance of circadian rhythms in synchronizing endogenous and behavioral systems, is crucial for proper neonatal and infant development and deserves further thorough investigations.

Tryptophan levels after chronotherapeutic Forest Environment trial or Vital Sky light or Standard Care on the ICU after cardiac surgery

Susanne Bengesser, Johanna M. Gostner, Michael J. Zwerger, Tatjana Stross, Rene Pilz, Miksch E, Andrea Pfeiffer, Paul Skacel, Nina Dalkner, Eva Reininghaus, Edita Lukic, M Schörghuber, Jolana Wagner- Skacel J & ICU Pflegeteam (Graz, Austria)

Department of Medical Psychology, Psychosomatics and Psychotherapy, Department of Psychiatry and Psychotherapeutic Medicine and Department of Anestesiology and Intensive Care, Medical University of Graz, Graz, Austria (susanne.bengesser@medunigraz.at)

All organisms on earth, from single cells to humans, had to adapt to the rotation of the earth and the challenges of the day night circle. An extraordinary clock gene circle has developed over time and orchestrates important body functions such as heart beat, blood pressure, hormone release – most important melatonin. The precursor tryptophan is therefore relevant for disturbed circadian rhythms, which are commonly distributed on Intensive Care Units (ICUs). To improve chronobiological challenges on the ICU after heart surgery, we applied three interventions to stressed ICU patients and in this analysis we focused on the melatonin precursor tryptophan.

The Forest Environment FFG trial included three groups receiving a chronotherapeutic intervention. The first group included patients after heart surgery, who received a „forest environment“ (from sunrise till sunset) via beamer on the ICU (n= 20). The second group included post-operative patients with standard ICU care as control group. The third group (n= 20) included post-operative patients with “Vital Sky ceiling”, which received light with 1900 LUX that is permanently installed on the ICU in certain rooms. Blood was taken at 3 study visits (day 1, day 3, day 7) at two time-points (22h p.m. and 5h a.m.) to measure the melatonin precursor tryptophan. The whole study protocol also includes NMR in serum, clock gene expression with qPCR in PBMCs from PAX gene tubes, melatonin and Tryp/Kyn markers, as well as psychological questionnaires.

Preliminary results point toward shifts in the Tryptophan pathway, but not all results are currently available. The results of the whole study will be presented at the next Pteridines workshop.

Development of new fluorescent Iridium (III) complexes as promising agents in photodynamic therapy

Elisabeth Quraishi, Bingjie Gao, Seah Ling Kuan, Tanja Weil, Till Opatz

Department of Chemistry, Johannes Gutenberg University Mainz, Germany (quraishi@uni-mainz.de)

Photodynamic therapy (PDT) is a minimally invasive treatment that combines light, photosensitizers (PS) and oxygen to generate cytotoxic reactive oxygen species (ROS) for the targeted destruction of diseased tissues, including cancers. PDT offers high spatial precision due to its localized light activation, minimizing systemic side effects. Metal complexes have emerged as highly promising photosensitizers in PDT, offering unique photophysical and photochemical properties that distinguish them from traditional organic PS. Their tunable electronic structures enable strong absorption in the therapeutic window (600–800 nm) for one-photon irradiation and, in some cases, allow two-photon absorption in the near-infrared region. This two-photon absorption capability is particularly advantageous, as it enables deeper tissue penetration and greater precision in targeting tumor sites. Additionally, metal complexes derived from transition metals or lanthanides exhibit enhanced ROS generation, photostability, and prolonged excited-state lifetimes, which are critical parameters for achieving therapeutic efficacy.

Our work focuses on the development and application of a novel class of Ir(III)-based photosensitizers, which can be easily functionalized with cell structure-specific targeting groups. Labeling photosensitizers enables versatile modifications, allowing for tailored applications, while maintaining their ability to target subcellular organelles without significant decline in targeting efficiency. Our studies demonstrated that these complexes co-localize in mitochondria, a key organelle for apoptosis induction. This mitochondrial localization significantly amplifies the photodynamic effect, as the production of ROS in close proximity to mitochondria triggers efficient cell death pathways. We have also shown that our new PS exhibit potent cytotoxicity against MDA-MB-231 triple-negative breast cancer cells, achieving promising therapeutic outcomes. Notably, their photodynamic activity is effective under both one-photon and two-photon irradiation. This versatility makes them well-suited for applications in both surface-level and deeper-seated tumors, addressing a critical limitation of traditional PDT agents. Mechanistic studies have provided insight into their efficient ROS generation, photostability, and selective accumulation in cancer cells.

These developments highlight the potential of Ir(III) complexes to overcome current limitations of PDT, such as insufficient tissue penetration, off-target effects, and low therapeutic selectivity. By combining photostability, organelle-specific targeting, and localized ROS generation, these next-generation PS offer a new platform for more effective and versatile PDT treatments. Our work provides a foundation for future functionalization of Ir(III)-based photosensitizers, emphasizing their role as transformative agents in advancing PDT and expanding therapeutic horizons for cancer and other diseases.

Virtual screening of pesticides for potential Janus kinase inhibition

Florian Fischer, Veronika Temml, Daniela Schuster

Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Paracelsus Medical University, Salzburg, Austria; Institute of Pharmacy and Research and Innovation Center for Regenerative Medicine and Novel Therapies, Paracelsus Medical University, Salzburg, Austria (florian.fischer@pmu.ac.at)

Background: Pesticides play a vital role in agriculture by protecting crops and boosting productivity, but their extensive use poses health risks. Farmworkers are directly exposed through skin contact and inhalation, potentially leading to hormonal imbalances, neurological disorders, and cancer. Residues in food and water can also affect surrounding communities. This work used pharmacophore modelling and virtual screening to identify pesticides that may inhibit Janus kinases (JAK1, JAK2, JAK3) and tyrosine kinase 2 (TYK2), critical for immune regulation but associated with severe side effects such as cancer and increased infection risk.

Methods: Pharmacophore modelling, a key technique in drug discovery, creates 3D representations of the essential physicochemical features responsible for biological activity. This in silico approach is commonly used in virtual screening to identify potential drug candidates. In this study, a combination of structure- and ligand-based modeling was used. These models were used to virtually screen the LUXPEST database of 386 pesticides to identify potential JAK inhibitors, after a theoretical evaluation process. The identified pesticides were cross-referenced with the Human Metabolome Database to determine whether these compounds have been detected in the human body.

Results: A total of eight models for JAK1, ten for JAK2, ten for JAK3, and nine for TYK2 were developed and optimized to cover over 90 % of their respective training sets. Screening the LUXPEST database identified fifty-eight pesticide candidates, with seventeen compounds, approved by the European Commission (EC) or the United States Environmental Protection Agency (EPA), detected in the human body. Examples include the herbicide cyanazine, which features a 1,3,5-triazine substructure, and sulfonylurea herbicides like tribenuron-methyl. These findings underscore the need for further research into their immunotoxic and chronic health effects.

Discussion: The biological evaluation of the identified pesticides provides experimental validation of the pharmacophore models, confirming their predictive accuracy and robustness.

Kinase inhibitors induce off-target effects on mitochondrial bioenergetics in cancer cell models

Sophie Strich, Andreas Feichtner, Selina Schwaighofer, Carolina Doerrier, Sabine Schmitt, Erich Gnaiger, Ludger Hengst, Eduard Stefan, Omar Torres-Quesada

Institute of Medical Biochemistry, Medical University of Innsbruck, Innsbruck, Austria; Tyrolean Cancer Research Institute (TKFI), Innsbruck, Austria (omar.torres-quesada@i-med.ac.at)

Protein kinases are critical regulators of numerous signaling pathways, including those controlling cell proliferation, differentiation, cell death, and metabolism. Dysregulation of kinase activity has been implicated in various human diseases, including cancer. Over recent years, kinase inhibitors have gained prominence as therapeutic agents targeting single or multiple oncogenic kinase pathways, evidenced by the approval of 49 kinase inhibitors by the FDA. Alongside kinases, mitochondrial metabolism has also emerged as a key therapeutic target. Mitochondria are dynamic organelles that play central roles in energy production and cellular signaling, and kinase activity blockade has been shown to impact mitochondrial function.

In our study, we employed targeted pharmacological interventions to manipulate kinase pathways implicated in mitochondrial function and oncogenesis. Using high-resolution respirometry, we systematically evaluated the effects of both broad-spectrum and specific kinase inhibitors on mitochondrial respiration in various cancer cell models. Our findings revealed that the impact of kinase inhibitors varies based on the mutational background of cancer cell lines and the composition of cell culture media.

Firstly, we observed off-target effects of sunitinib, a multikinase inhibitor approved by the FDA, only in a more physiologically relevant cell culture medium compared to classical formulations. Secondly, mitochondrial profiling of the glycolytic kinase inhibitor PFK158 demonstrated off-target mitochondrial dysfunction. Thirdly, we showed that kinase signaling inhibition is linked to mitochondrial reactive oxygen species (ROS) production, which can be modulated by kinase-targeting drugs.

In summary, our findings highlight the potential of mitochondrial bioenergetic profiling to uncover drug-induced mitochondrial dysfunction and off-target effects of kinase inhibitors. This approach offers valuable insights for predicting drug effects on cell metabolism and underscores the utility of cell-based diagnostics in therapeutic development.

Biopsychronology 2.0: Taking a closer look at blood of Long-COVID patients

Martin Hermann, Christoph Lisch, Regine Gerth, Tatjana Marth, Georg Wick, Katharina Kurz, Dietmar Fries, Nikolaus Wick

Department of Anesthesia and Intensive Care, Medical University Innsbruck, Austria; Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria; Sanatorium Hochrum, Austria; Specialized Laboratories Wick, Innsbruck Austria; Fachhochschule für Gesundheit, Innsbruck, Austria; Division of Pathophysiology, Biocenter, and Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria. (martin.hermann@tirol-kliniken.at)

Patients who recovered from COVID-19, but are still experiencing symptoms such as shortness of breath, cognitive dysfunction (brain fog) or chronic fatigue, suffer from a condition known as “Long-COVID”. The underlying nature of this illness still remains well hidden behind it ́s plethora of signs and symptoms, making it difficult to find a specific treatment option. Herein we describe an imaging approach, which promises to shed more light and tell us more about what is going on.

In the past, we developed an approach referred to as biopsychronology, which combines the use of indicator dyes for cellular function and integrity with live cell confocal imaging of solid biopsies to gain rapid insight into the functional status of tissues and organs prior to transplantation. Herein, we apply this method using liquid biopsies i.e. a drop of blood of long-COVID patients, incubated under static conditions with live stains such as

Methods: HOECHST, Wheat-Germ Agglutinin and ffbp (fibrin binding protein) in order to visualize nuclei, cell morphology and fibrin/microclots.

We were able to get a grip on the heterogenic nature of long-COVID applying the biopsychronology method. Each patient ́s blood tells its story. We were able to visualize live in real time differences in cell morphology, the presence of microclots, numbers of granulocytes, NET formation, platelet interactions, Rouleaux formation, as well as structures, which we named Long COVID Plaques.

The basis for every treatment is the understanding of the underlying problem. Our live confocal imaging approach provides us live in real time with a picture of our blood reacting to challenges such as long-COVID. Due to the incubation under static conditions, we have a chance to get a glimpse of what is going on in each individual blood sample. Our data already provide the basis for a successful therapy in LC patients.

Therapeutic efficacy of inhaled N-chlorotaurine in a mouse model of Aspergillus fumigatus Pneumonia

Cornelia Speth, Günter Rambach, Andrea Windisch, Nadine Falbesoner, Christoph Schatz, Georg Schäfer, Markus Nagl

Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria (m.nagl@i-med.ac.at)

Recently, we could demonstrate the efficacy of the endogenous antiseptic N-chlorotaurine (NCT) applied via inhalation in Lichtheimia corymbifera pneumonia in the mouse. In the present study, this was shown in the mouse model of Aspergillus fumigatus pneumonia.

Specific pathogen-free female C57BL/6JRj seven-week old mice were immune-suppressed with cyclophosphamide or cortisone acetate. After 7 days, they were inoculated intranasally with 6.5 x 10E6 spores of A. fumigatus, i.e. a slightly lower count than in a previous study where it was too high with unclear outcome. Inhalations with 0.1%, 0.5%, 1.0% (55 mM) or 2.0% NCT solution or 0.9% sodium chloride three times daily for 10 min started one hour after inoculation and ended after 15 days.

In mice euthanized on day 2 in all groups (n = 5), fungi in the lung and other signs of pneumonia were found. In the placebo group, 8/9 or 9/9 mice observed for 15 days died from the infection during this time, while 0/9 to 1/9 died in groups treated with 0.5%, 1.0% and 2.0% NCT (p < 0.01 for each concentration versus saline). There was no difference between the two ways of immune-suppression. With 0.1% NCT, 4/9 mice died (p = 0.03 versus the higher NCT-concentrations; p = 0.0035 versus control). The fungal load came to 5.28 (4.46; 5.70; median, quartiles) colony-forming units per ml lung homogenate in the control group and to 1.3 (median; maximum 2.45) in the 1% NCT group in mice immune-suppressed with cyclophosphamide (p = 0.0004). Values were similar in cortisone groups (p = 0.0023). Secondary parameters showed respective significant differences between test and control groups.

Early treatment with inhaled NCT demonstrated highly significant efficacy in Aspergillus pneumonia, too. A concentration of 1% NCT appears to be optimal, which fits to case experiences with inhalations in humans.

Urine metabolome analyses, inflammation parameters and other potential biomarkers in patients with post-acute infection syndromes (PAIS)

Katharina Kurz

Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria Lanserhof Medical Spa, Lans, Austria (Katharina.Kurz@i-med.ac.at)

Post-acute infection syndromes (PAIS) like post-covid syndrome (PCS) and myalgic encephalitis/chronic fatigue syndrome (ME/CFS) are an increasing health problem. After Covid 19-infection or other infections like Epstein Barr virus infection, but also after Covid-19 vaccination, patients can suffer from persistent symptoms over months or years. The most disabling ones are fatigue (often with post-exertional malaise), neurological symptoms like brain fog and concentration/memory deficits, sleep disturbances, pain and symptoms of dysfunction of the autonomic nervous system. In this pilot study we did urine metabolome analyses and blood analyses in patients with PAIS, analyses were performed by Biovis Diagnostics (Limburg, Germany).

While standard lab parameters were mostly within reference ranges, urine metabolome analyses and auto-antibody analyses frequently showed conspicuous results: Urine metabolome analyses often showed reduced concentrations of amino acids (tryptophan, phenylalanine, tyrosine) and/or their metabolites (serotonin, kynurenine, kynurenic acid, quinolinic acid, NAD; dopamine, adrenaline, noradrenaline). Concentrations of bacterial uremic metabolites were found in the majority of patients- indicating that gut dysbiosis is an important pathomechanism. Also, antibodies against G-protein coupled receptors (GPCR-antibodies) were elevated in many patients, also other antibodies were often positive (especially IgG for IL-6 or IL6R). Inflammation parameter serum calprotectin was nearly always too high, whereas Th1 type immune activation marker neopterin was elevated only in few patients. Markers of oxidative and nitrosative stress, which are induced by overactivation of the immune system, were elevated in about half of the patients. Lactate dehydrogenase (LDH) isoenzymes as screening markers of secondary mitochondrial dyfunction were often outside the normal range. Lytic microaggregates (detected by confocal microscopy) were found in a significant percentage of patients. In addition, concentrations of pregnenolone (as precursor of steroid hormones) were frequently low, especially in perimenopausal women.

Conclusively, data of our pilot study with precision medicine diagnostics show that in patients with PAIS urine metabolome analyses and special blood tests (like GPCR-antibodies, pregnenolone) and microaggregate analysis often show conspicuous results, and that in every patient a combination of different pathomechanisms appears to be underlying. The most important pathomechanisms underlying PAIS appear to be gut dysbiosis, dysregulated immune response, neurotransmitter dysbalance, hormon dysbalance and mitochondrial dysfunction due to enhanced oxidative/nitrosative stress. Further research is essential to characterize the above-mentioned pathomechanisms in more detail in PAIS and evaluate personalized treatment approaches.

Metabolome analyses in patients with post-acute infectious syndromes (PAIS)

Katharina Wagner

University Clinic for Internal Medicine II, Infectiology, Innsbruck, Austria (Katharina.Wagner@i-med.ac.at)

Post COVID syndrome (PCS) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are complex conditions that affect multiple body systems, causing debilitating symptoms. Around 10% of people recovering from COVID-19 experience persistent symptoms like fatigue, breathlessness, and cognitive or psychological problems. Routine laboratory tests often show inconspicuous results, making it hard to identify the underlying mechanisms: This pilot study aims to explore whether metabolic changes can be detected by urine and blood plasma metabolome analyses in patients with PCS and ME/CFS.

Urine metabolome and blood plasma analyses were performed in patients with PCS, healthy controls (Ctrls), individuals with ME/CFS as well as individuals with long-term side effects after COVID-19 vaccination (Post-Vac; PV). Mass spectrometry was employed for analyses. The concentrations of neurotransmitter precursors tryptophan, phenylalanine, and their downstream metabolites in urine and blood plasma were analyzed.

Urine metabolome showed that phenylalanine was higher in the Ctrl group than in the PCS and ME/CFS group. Serum glutamate levels were significantly higher in the ME/CFS group compared to both the PV group and the Ctrl group. Additionally, neopterin levels in blood were higher in the PCS group than in the ME/CFS group. Gender differences were also observed in the analysis: Serum glutamate levels were lower in women compared to men, while urinary glutamate levels were higher in women than in men.

This study shows metabolic disturbances in PCS and ME/CFS: Dysbalances in amino acid and neurotransmitter metabolism, as well as immune activation. Findings of our pilot study are promising, as new biomarkers for PCS and ME/CFS are urgently needed. Further studies focusing on the above-mentioned metabolic pathways (such as glutamate signaling, immune activation, and amino acid metabolism) are needed to better understand and treat LC and CFS.

Pilot study of autoantibodies in patients with Long Covid

Peter Rappl, D. Neupärtl, Burkhard Schütz, Samuel Gonos, Sabine Engl, Judith Löffler-Ragg, Günter Weiss, Katharina Kurz

Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria (samuel.gonos@i-med.ac.at)

The impact of SARS-CoV-2 has resulted in millions of infections worldwide, with patients developing persistent symptoms known as post-COVID syndrome (PCS) or long COVID. The reactivation of Epstein-Barr virus (EBV) and autoimmune-driven autoantibody production are among the suspected pathomechanisms. Within this pilot study, several autoantibodies from the IgA and IgG groups (Prothrombin, Beta2GPI, MPO, proteinase3, IL-6, IL-6R, ACE2, and GM-CSF) as well as EBV-specific antibodies were determined to better understand the potential role of these autoantibodies and EBV-reactivation in PCS.

Antibodies were determined in 311 PCS patients and 238 healthy controls (HC). There were significant differences between PCS and HC: IgA MPO, IgA Proteinase 3, IgG Beta2-GPI, and IL-6R antibodies were found significantly more often in PCS compared to HC. Correlation analysis revealed associations between autoantibodies in PCS patients, especially between IgA MPO and IgA Proteinase 3 as well as IgG Beta2-GPI and IL-6R IgG.

Findings of our pilot study indicate distinct autoantibody profiles in PCS compared to HC, which might be useful to better diagnose distinct subtypes of PCS and thus, enable personalized treatment in these patients.

Iron and infection. New insights.

Iana Portnaia, Alexander Hoffmann, Egon Demetz, Chiara Volani, Markus Seifert, Günter Weiss

Department of Internal Medicine II, Infectious Diseases, Immunology, Rheumatology, Pulmonology, Medical University of Innsbruck, Innsbruck, Austria (iana.portnaia@i-med.ac.at)

Bacteria rely on iron for metabolic processes, and during infection, pathogens compete with the host for this critical resource. This study focuses on Salmonella Typhimurium (S. Typhimurium), a facultative intracellular gram-negative bacterium that resides in macrophages within the Salmonella-containing vacuole (SCV). S. Typhimurium evades the host’s nutritional immunity to utilize host-derived iron for growth. Its replication within macrophages depends on iron availability in the SCV, making it important to understand how it acquires and uses iron sources.

To examine the effects of iron supplements on bacterial growth, we measured doubling times at varying iron concentrations using the absorbance method. Two iron delivery methods were compared: oral supplementation and intravenous (i.v.) administration. Oral iron is absorbed in the duodenum and circulates extracellularly, while i.v. iron compounds (e.g., Ferric-Isomaltoside 1000, Ferric Carboxymaltose) form carbohydrate complexes, leading to intracellular iron accumulation. Oral iron, being more accessible to bacteria, may worsen infections.

Ferric nitrate nonahydrate (FeN9H) and ferric maltol (FM) significantly reduced bacterial doubling times, with a 2-fold reduction observed at 25 µM, commonly used in in vitro studies. Conversely, i.v. iron was less accessible to S. Typhimurium. Using RAW 264.7 macrophages infected with a fluorescently tagged S. Typhimurium strain, flow cytometry showed reduced iron starvation signals (via the BFP[RyhB2] reporter) in media supplemented with FeN9H and FM, indicating greater iron accessibility compared to i.v. sources.

Bacterial load was assessed by calculating colony-forming units (CFUs) 24 hours post-infection with wild-type S. Typhimurium (ATCC 14028). No significant differences were observed across treatments, likely due to delayed cell collection and cells mortality.

PCR analysis was used to evaluate the immunomodulatory effects of bacterial supernatant. Supernatant from S. Typhimurium cultures grown in LB broth (2-hour incubation, filtered at 0.22 µm) was applied to peritoneal macrophages (PMs) without washing with phosphate-buffered saline and ensuring the absence of bacteria. The supernatant induced inflammatory gene expression, promoting M1 macrophage polarization after 4 hours of inoculation. IL-6 expression increased in a dose-dependent manner with higher supernatant concentrations.

As a facultative intracellular pathogen, S. Typhimurium is not fully eliminated by gentamicin, potentially affecting CFU results. Live-cell imaging revealed extracellular bacteria, possibly forming biofilms. The role of biofilm formation and bacterial release during macrophage lysis in infection progression warrants further investigation.

Deletion of tumour necrosis factor in mice leads to promotion of Salmonella enterica serovar Typhimurium multiplication by modulating macrophage iron homeostasis.

Natascha Brigo, Christa Pfeifhofer-Obermair, Piotr Tymoszuk, Philipp Grubwieser, Sabine Engl, Markus Seifert, Dirk Bumann, Ulrike Schleicher, Christian Bogdan, Günter Weiss

Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria (natascha.brigo@i-med.ac.at)

Tumour necrosis factor alpha (TNF) is crucial for controlling intracellular bacterial infections and affects cellular iron homeostasis by modulating the iron storage protein ferritin (FTH) and the cellular iron exporter ferroportin-1 (FPN-1), thereby affecting cellular iron levels. This impacts intracellular multiplication of siderophilic bacteria like Salmonella enterica serovar Typhimurium (S.tm), influencing infection control.

To study TNF’s impact on Salmonella infection, we examined bone marrow-derived macrophages (BMDM) from TNF-/- mice and observed a significantly reduced S.tm killing capacity compared to wild-type BMDM, accompanied by reduced FPN-1, increased FTH expression and expanded intracellular iron levels along with diminished antimicrobial immune responses (iNOS, NO, IL-1). TNF-/- mice succumbed significantly faster to infection than wild-type mice, which was paralleled by increased bacterial numbers in the spleen, liver and blood. Whole-body iron homeostasis analysis showed reduced FPN-1 expression and iron accumulation in the spleen and liver, facilitating bacterial access to iron as confirmed by employing iron-sensitive fluorescence plasmids in the bacterial strain.

Mechanistically, TNF deletion increased IL-6 formation, inducing hepcidin transcription in the liver. Hepcidin leads to proteolytic degradation of FPN-1 and blocking of iron export. Treating TNF-/- BMDM with the iron chelator deferasirox (DFX) improved infection control and increased anti-microbial immune effector pathways. Data from rheumatoid arthritis patients receiving anti-TNF treatment also indicated increased hepcidin and serum iron levels three months post-treatment.

Thus, TNF deletion alters iron metabolism, leading to iron retention through hepcidin-mediated blockade of iron export. This increases iron availability to intracellular S.tm, promoting bacterial proliferation and impairs infection control.

PCBP2-dependent iron trafficking controls Salmonella infection in macrophages

Manuel Grander, David Haschka, Günter Weiss

Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria (manuel.grander@i-med.ac.at)

Upon infection with siderophilic pathogens such as Salmonella, macrophages are required to tightly control their iron homeostasis to limit pathogen survival. The iron chaperone PCBP2 is considered to be crucial in intracellular iron distribution, but its role in host defense remains unclear. This study investigated the impact of PCBP2 in the control of Salmonella infection of macrophages.

To assess the impact of PCBP2 on Salmonella infection, we utilized bone marrow derived macrophages with a targeted knockout of PCBP2. We employed fluorescent iron-sensing Salmonella to measure local iron availability, applied iron chelators and iron uptake-deficient Salmonella mutants to determine the impact of iron in Salmonella proliferation. Iron distribution was analyzed through various measurements of labile and total iron pools, while RNA sequencing was performed to identify key pathways regulated by PCBP2 upon infection.

PCBP2 knock out (KO) macrophages exhibited impaired clearance of Salmonella Typhimurium. Infection with fluorescent iron-sensing Salmonella revealed that PCBP2 restricted iron availability to Salmonella. The survival advantage of Salmonella in PCBP2 KO macrophages was abrogated by the addition of iron chelators or the use of iron uptake-deficient Salmonella, indicating an iron-dependent mechanism. Mechanistically, PCBP2 KO macrophages showed altered iron distribution with reduced labile iron but elevated total iron levels. Infection-induced macrophage iron depletion was diminished in PCBP2 KO cells. RNA-seq analysis highlighted significant upregulation of lysosomal pathways in infected PCBP2 KO macrophages. Furthermore, the use of transferrin as an endo/lysosomal iron source markedly enhanced Salmonella iron acquisition and proliferation, while endolysosomal iron export was notably reduced in PCBP2-deficient macrophages.

Our findings demonstrate that PCBP2 plays a pivotal role in controlling intracellular iron trafficking during Salmonella infection, facilitating iron sequestration and limiting pathogen iron access. The impairment in iron homeostasis observed in PCBP2-deficient macrophages underscores its critical function in macrophage-mediated defense mechanisms against siderophilic pathogens.

Unraveling tryptophan’s fate in glioblastoma metabolism

Francisco Yanqui-Rivera

DKTK Metabolic Crosstalk in Cancer, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), and Faculty of Bioscience, Heidelberg University, Heidelberg, Germany (f.yanquirivera@dkfz.de)

Abstract not sent in time

The tryptophan-kynurenine pathway- therapeutic strategy for neuroprotection in tauopathies

Krutika Khiratkar, Petra Majerova, Andrej Kovac, László Vécsei, Viktor Milata, Kevin James

Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovakia (krutika.khiratkar@savba.sk)

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that deteriorates cognitive functions, characterized by the intracellular accumulation of abnormal filaments of the microtubule-associated protein tau, amyloid-β plagues (Aβ), and neuronal apoptosis in the central nervous system (CNS) releasing pro-inflammatory cytokines with hyper-reactive brain immune cells (microglia and astrocytes). Moreover, these deposits are associated with systemic inflammation and disruption of the many signaling pathways including the kynurenine pathway. The tryptophan-kynurenine pathway is functionally maintained by the catabolism of the essential amino acid, L-tryptophan. This pathway generates various metabolites specifically the kynurenic acid (KA), a neuroprotective metabolite in the CNS. In neuroinflammatory conditions, under the influence of inflammatory cytokines (IFN-γ) and reactive oxygen species (ROS), a key regulatory enzyme indoleamine 2,3-dioxygenase (IDO) is induced, declining the neuroprotective metabolite levels and increasing the production of a neurotoxic agent such as quinolinic acid (QA). The IDO-induced QA over-stimulates the excitatory receptor N-methyl-D-aspartate (NMDA) acting as an agonist whereas, KA acts as an antagonist of this receptor modulating the functioning of the brain. Therefore, we aim to screen analogs of kynurenic acid having similar biological activity along with higher blood–brain barrier (BBB) permeability, to modulate the neuroinflammation in the CNS and its functions using an in-vitro BBB model as well as an in-vivo model using the transgenic rats. Our screening of various analogs identified few compounds with both high blood-brain barrier permeability and promising biological activities. These findings suggest potential for effective treatment of neuroinflammatory conditions.

Kynurenine pathway, neopterin, heavy metals and trace elements in electronic waste recycling industry workers

Sara Muhammetli, Canan Demir, Sonia Sanajou, Gözde Girgin, Terken Baydar

Department of Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turky; Department of Occupational Medicine, Faculty of Medicine, Dokuz Eylul University, Izmir, Turky (drcanandemir18@gmail.com)

Many environmental and occupational exposures may affect the immune system and can potentially disturb homeostasis in human organisms. Since lead (Pb), cadmium (Cd), and antimony (Sb) are widely used in electronic devices, recycling processes can release significant amounts of those metals along with other toxic substances, and combined exposure is highly possible. The hypothesis that the inhalation of heavy metals during the recycling process disrupts trace element homeostasis, may have effects on cellular immune response, changes neopterin levels, and alters the kynurenine pathway constitutes the main goal of the study performed on e-waste recycling industry workers.

Thirty male volunteers working in the e-waste recycling industry were included. Detailed occupational anamnesis and physical examination were performed for each participant. Blood samples were collected for biochemical and toxicological tests. Essential trace elements chromium (Cr), cobalt (Co), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se), and zinc (Zn), and heavy metal levels (Sb, Cd, and Pb) were quantified using the inductively coupled plasma-mass-spectrometry (ICP-MS) technique. Serum tryptophan (Trp) and kynurenine (Kyn) levels were determined using verified high-performance liquid chromatography (HPLC). Serum neopterin levels were quantified using a commercially available ELISA kit (IBL, Germany). IBM SPSS 23 statistical software was used for data analysis. Variables were presented as mean and standard deviation (SD) /error (SEM). Homogeneity was evaluated with Levene’s Test. The alpha value was accepted as 0.05.

No statistically significant relation was found between the symptoms of the participants and elevated metal levels. Serum neopterin, Trp, and Kyn concentrations were 7.8 ± 0.7 nmol/L, 54 ± 2 µmol/L, and 0.5 ± 0.04 µmol/L, respectively. The estimated indoleamine 2,3-dioxygenase (IDO-1) activity was calculated as 8.8 ± 0.8 µmol/mmol. Blood lead levels were positively correlated with antimony, and neopterin was negatively correlated with copper.

The enhanced Pb burden and metals such as Sb, Cd, Ni, Mn, and Co indicate the multiple metal exposure of e-waste workers. Those increased metal levels were not related to any changes in trace element status, neopterin release, and Kyn pathway. Further studies with increased numbers of workers concerning multiple metal exposures should be conducted to establish and understand the underlying toxicological mechanisms and health effects. Despite we cannot establish a significant relationship between high metal levels and neopterin levels, and the kynurenine pathway in this study, the e-waste recycling industry continues to be a considerable exposure source for workers, their families, residents, and the environment. For decreasing the exposure levels and primary protection concerns; effective and comprehensive measures should be taken.

From senescence to bioactivity: Exploring phyllobilins in Papaya leaves

Christian Elvert, Rao Zhigang, Johanna Gostner, Andreas Koeberle, Fabian Hammerle, Cornelia Karg, Awodayo O Adepiti, Simone Moser

Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Center for Chemistry and Biomedicine (CCB), Austria (Christian.Elvert@uibk.ac.at)

Carica papaya leaf extracts (PLE) have a long history of traditional medicinal use in African countries, particularly for the treatment of inflammatory diseases. Notably, some communities in Africa prefer withered leaves that have fallen to the ground for the preparation of extracts, considering them more potent than extracts of fresh leaves. Phyllobilins are degradation products of the green plant pigment chlorophyll, formed primarily during senescence of the plant. So far, over 70 different phyllobillins from more than 30 plant species have been identified. Even though they are ubiquitous in nature, these breakdown products have largely been overlooked as bioactive compounds. Several studies revealed pharmacologically relevant effects of phyllobilins from different plants, including anti-oxidative properties, anti-proliferative effects on cancer cells as well as anti-inflammatory.

This study aims at characterizing extracts of Carica papaya leaves at various stages of senescence, with a focus on identifying and isolating bioactive phyllobilins, since phyllobilins are senescence associated compounds, we expect to find increased amounts of phyllobilins in senescent and withered leaves. Previous results revealed notable bioactivity across different phyllobilin structures, with pronounced anti-oxidative and anti-inflammatory effects, including an effect on tryptophan degradation in human peripheral blood mononuclear cells (PBMCs). These findings show the promise of studying phyllobilins in Carica papaya leaves.

Natural tetrapyrroles and cardiovascular health: Exploring the atheroprotective potential of bilirubin and phyllobilins

Benjamin Kirchweger, Alexandro P. Roggio, Giulia Verilli, Matthias Völkl, Cornelia Karg, Simone Moser, Robert Fürst

Pharmaceutical Biology, Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-Universität München, Germany (benjamin.kirchweger@cup.uni-muenchen.de)

Atherosclerosis-driven cardiovascular diseases are the leading global cause of mortality. Several epidemiological studies have observed that individuals with elevated serum bilirubin levels exhibit a degree of protection against atherosclerosis. Bilirubin, a degradation product of heme, is hypothesized to possess bioactive properties that may mediate these protective effects, though causality remains unconfirmed. Structurally similar tetrapyrroles, the phyllobilins, are generated during chlorophyll breakdown in plants and may also act as specialized metabolites. Phyllobilins are commonly found in herbal medicines and dietary sources and thus may contribute to health and disease prevention. This study aimed to investigate the effects of bilirubin and phyllobilins on key cellular processes of atherosclerosis.

To evaluate the potential roles of bilirubin and phyllobilins, a comprehensive array of functional in vitro assays was conducted to model cellular mechanisms associated with atherosclerosis. These experiments were performed using primary human cells, including human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells (HAoSMCs), and peripheral blood mononuclear cells (PBMCs), ensuring greater relevance to the clinical setting.

Results demonstrated that both bilirubin and phyllobilins exhibited significant bioactivity in processes relevant to atherosclerosis. Bilirubin and phyllobilins (i) displayed robust antioxidant effects, reducing oxidative stress in vitro and in living cells, which may mitigate lipid oxidation in vivo. (ii) They inhibited collagenase activity, potentially limiting the remodelling of the atherosclerotic vessel wall. (iii) Bilirubin significantly reduced the uptake of oxidized low-density lipoprotein (oxLDL) by human macrophages, a critical step in foam cell formation. (iv) One phyllobilin showed significant inhibition of vascular smooth muscle cell proliferation and migration, processes central to the progression of atherosclerotic plaques.

These findings suggest that bilirubin and phyllobilins exhibit bioactive properties in processes associated with atherosclerosis. The observed effects indicate that these tetrapyrroles offer a potential avenue for therapeutic exploration.

Niche techniques in modern pharmacognosy – taking FCPC as an example

Michael J. Zwerger, Johanna M. Gostner, Simone Moser, Markus Ganzera

Institute of Medical Biochemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria; Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Innsbruck, Austria (michael.zwerger@i-med.ac.at)

Modern phytochemistry strongly relies on modern and efficient analytical and preparative techniques. Apart from routine assays, also more “exotic” methodologies have been developed, offering unique advantages in term of economy and orthogonal selectivity. A good example in the field of chromatography is fast centrifugal partition chromatography (FCPC). Here, the separation of the analytes is based on a system of two immiscible liquid phases, thus requiring no solid stationary support. The (liquid) stationary phase is held in the instrument by centrifugal forces while the mobile phase passes through Therefore, possible sample loss due to irreversible binding to the stationary phase is prevented. The purification and separation of compounds is achieved solely by their different partition coefficients in the upper and lower phase, respectively. FCPC can be used for an array of natural compounds, of which two examples are presented: alkaloids from Chelidonium majus and mycosporine-like amino acids from various red algae, whereby established approaches could be surpassed by this emerging technology. Considering the challenging isolation of phyllobilins, FCPC also seems a promising option in the field of future bilin-related research.

Tetrahydrobiopterin: A crucial mediator in colitis-associated abdominal pain

Ananda Staats Pires, Bruna Lenfers Tuner, Débora da Luz Scheffer, Vivian de Souza Menegassi, Sara Marques Rizzattii, Laís Niero, Gilles J. Guillemin, Alexandra Latini

Neuroinflammation Group, Department of Biomedical Sciences, Centre for Motor Neuron Disease Research, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia; Laboratório de Bioenergética e Estresse Oxidativo, Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianopolis, Brazil (AnandaChristina.StaatsPires@med.uni-jena.de)

Ulcerative colitis (UC) is a chronic inflammatory disorder of the colonic mucosa characterized by alternating periods of exacerbation and remission. Abdominal pain is a prevalent symptom during the course of UC and persists in approximately 30% of affected patients, even in the absence of endoscopic lesions or markers of active inflammation. This observation suggests that factors beyond inflammation may contribute to the symptomatology of UC-related abdominal pain.

Tetrahydrobiopterin (BH4) is a cofactor traditionally studied for its role in the synthesis of neurotransmitters (e.g., serotonin) and nitric oxide. Recent research has highlighted the excessive activation of the BH4 pathway in various models of inflammatory and neuropathic pain, suggesting its potential involvement in pain mechanisms.

The aim was to investigate whether activation of the BH4 pathway contributes to abdominal pain in UC.

A cross-sectional study was conducted involving patients with a clinical diagnosis of UC (n = 60) and age- and sex-matched healthy participants (n = 55). Participants rated their abdominal pain using a Visual Analogue Scale (VAS). Urine and blood samples were collected for biochemical analyses.

Patients with UC experiencing abdominal pain exhibited elevated levels of BH4 in both urine and plasma compared to healthy participants. Furthermore, urinary BH4 levels were positively correlated with plasma serotonin levels, which in turn showed a positive correlation with the severity of abdominal pain.

These findings suggest that altered BH4 biosynthesis may influence serotonin availability, contributing to abdominal pain in UC. This research underscores the need for further studies to identify the specific sources and mechanisms of BH4 activation in colitis-associated abdominal pain.

In silico study of natural antioxidants

Andrey Buglak

Saint Petersburg State University, St. Petersburg, Russia (andreybuglak@gmail.com)

Antioxidant properties of various compounds can be estimated using theoretical chemistry and three single electron oxidation mechanisms: 1) hydrogen-atom transfer (HAT); 2) single electron transfer followed by proton transfer (SET-PT); and 3) sequential proton-loss electron transfer (SPLET). Amino acids (AAs) are the building blocks of life, and some of them possess antioxidant properties.

Antioxidant properties of 21 proteinogenic amino acids and 3,4-dioxophenylanine (DOPA) were evaluated in aqueous media using density functional theory (DFT). Five most reactive AAs have been established: DOPA, selenocysteine (Sec), tyrosine (Tyr), cysteine (Cys) and tryptophan (Trp). Also, global reactivity in terms of hardness/softness has been evaluated, as well as Fukui indices of local reactivity. Trp has been determined as the most reactive molecule, whereas catechol ring oxygen of DOPA and selenium atom of Sec have been established as the most reactive atoms.

All the findings are in agreement with the recent literature on both experimental and theoretical studies of amino acids antioxidant activity; thus, AAs can be used for treating oxidative stress and oxidative stress-related diseases: cancers, vitiligo, etc. Moreover, other classes of antioxidant molecules can be measured using this methodology: pterins, alkaloids, stilbenes can be mentioned to name a few.

Activation of IDO-1 is the key event that drives immunotoxicity

Lucia Parráková, Oriol Ruiz Catalan, Pablo Monfort-Lanzas, Dietmar Fuchs, Johanna M Gostner

Institute of Medical Biochemistry, Biocenter, Medical University Innsbruck, Austria (lucia.parrakova@i-med.ac.at)

Indoleamine 2,3-dioxygenase 1 (IDO-1) is an enzyme expressed in antigen-presenting cells and induced by IFN-γ during a Th1-type immune response. It is the rate-limiting enzyme in the degradation of tryptophan into kynurenine. This conversion has two immunological effects: depletion of tryptophan, which restricts the proliferation of pathogens, and the production of kynurenine and its metabolites, which act as immunomodulators.

IDO-1 activity, typically measured as the ratio of kynurenine to tryptophan in cell culture supernatants, serves as an indirect marker of immune activation and can be exploited in vitro to evaluate the immunomodulatory effects of chemical compounds.

In this study, we investigated the effect of Bisphenol A (BPA), a known endocrine disruptor with suspected immunotoxic effects, and six of its analogues (TCBPA, BPE, BPAP, BPZ, BPP, and BPS-MAE) on IDO-1 activity in human peripheral blood mononuclear cells (PBMCs). Cells were either stimulated or not with phytohaemagglutinin (PHA), a mitogen that induces a Th1-like immune response.

PBMCs were freshly isolated from healthy donors and treated in vitro with concentrations ranging from 12.5 to 200 μM of each bisphenol, in the presence or absence of PHA. Supernatants were collected after 24 and 48 hours of treatment and analyzed by high-performance liquid chromatography (HPLC) to quantify tryptophan and kynurenine levels. Cell viability was assessed in parallel using a resazurin reduction assay.

A dose-dependent reduction in IDO-1 activity was observed for all bisphenol analogues in stimulated cells at concentrations below cytotoxic levels, with more pronounced effects at 48 hours compared to 24 hours. IDO-1 activity in unstimulated cells was not significantly affected.

These results demonstrate that a PBMC model combined with IDO-1 activity measurement provides a viable in vitro system for assessing the immunomodulatory potential of chemical substances. This approach offers a cost-effective and ethically favorable alternative to animal models and allows for personalized assessments when using patient-derived cells.