Sample treatment for tissue proteomics in cancer, toxicology, and forensics

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science. This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies. In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution. With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies. Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest

MALDI-MS investigation of skin and its response to irritants and sensitisers.

There is increasing interest in in-vitro methodology for testing chemical toxicity, one of the drivers for this is European legislation; specifically Directive 76/768 EEC, which prohibits animal testing in the cosmetic industry. In skin toxicological testing, an alternative methodology to in-vivo irritancy experiments already available uses a synthetic skin model and involves measurement of cell viability and release of cytokine interleukin (IL)-1a. There is currently no fully validated in-vitro test for sensitization, although there are a number that are in development. Some of these include the human cell line activation test (h-CLAT) (Sakaguchi et al., 2010), the direct peptide reactivity assay (DPRA) (Gerberick et al., 2009) and the myeloid U937 skin sensitization test (MUSST) (Ade et al., 2006). This study utilises ex-vivo human skin as an initial platform for method development. Areas of analysis have included the lipidomic and proteomic responses of ex-vivo human skin to sensitizers and irritants.Matrix assisted laser desorption ionisation-ion mobility separation-mass spectrometry (MALDI-IMS-MS) was used to identify peptides and lipids in-situ. Ion mobility separation allowed for discrimination between isobaric species due to the selection of specific precursor ions and cleaner MS/MS spectra. Many of the peptides identified belonged to serum albumin, keratin and collagen protein families, which are known to be abundant within human skin. Lipids identified included: sphingomyelin, glycerophospholipids, ceramide species anddi/triglycerides.In skin studies performed in other research groups, sphingomyelin and ceramide species were found to change expression in response to initiation of inflammation, thus making them of biological significance when investigating lipidomic responses to chemical sensitizers and irritants. Tryptic peptide MALDI-MS images have shown differences in human skin, as a result of chemical exposure. MALDI-MS images at 150pm and 30pm resolution have provided information on the localisation of detected species. Changes in expression of species in treated and untreated samples, as well as between different layers of human skin have also been visualised via multivariate statistical analyses. These analyses of ex-vivo human skin have demonstrated that MALDI-IMS-MS is a technique which can be used to detect changes in protein/peptide and lipid profiles in response to sensitisers and irritants. The technique also allows for the localisation of species detected within the different layers of human skin

Towards Precision Dermatology: Emerging Role of Proteomic Analysis of the Skin

Background: The skin is the largest organ in the human body and serves as a multilayered protective shield from the environment as well as a sensor and thermal regulator. However, despite its importance, many details about skin structure and function at the molecular level remain incompletely understood. Recent advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics have enabled the quantification and characterization of the proteomes of a number of clinical samples, including normal and diseased skin. Summary: Here, we review the current state of the art in proteomic analysis of the skin. We provide a brief overview of the technique and skin sample collection methodologies as well as a number of recent examples to illustrate the utility of this strategy for advancing a broader understanding of the pathology of diseases as well as new therapeutic options. Key Messages: Proteomic studies of healthy skin and skin diseases can identify potential molecular biomarkers for improved diagnosis and patient stratification as well as potential targets for drug development. Collectively, efforts such as the Human Skinatlas offer improved opportunities for enhancing clinical practice and patient outcomes

Application of imaging MALDI-MS to the determination of hazardous compounds in skin.

The overall aim of this work described in this thesis was to apply MALDI-MS and imaging MALDI-MS (MALDI-MSI) to the analysis of compounds of interest in occupational hygiene monitoring.Isocyanates are highly reactive compounds with a wide variety of industrial uses. MALDI-MS and MS/MS were used to investigate diisocyanate stability and reactivity. A monoisocyanate intermediate product was observed from the hydrolysis of both an aromatic and also an aliphatic diisocyanate. The stability of this product was assessed over a 14 day experimental period. Ethanol was used to derivatise hexamethylene diisocyanate (HDI) and the resulting urethane was observed to be stable over 14 days. This derivatisation method was incorporated into a surface swab technique for sampling of HDI from work surfaces.Industrial diisocyanates have been reported to penetrate through skin and to be excreted as diamine metabolites in urine. A LC-MS method for the determination of free toluene diamine (TDA) monomer formed by biotransformation of toluene diisocyanate (TDI) when applied to HaCaT cells was developed. In the two experiments performed, TDA was only observed at low levels after spiking with high concentrations of TDI. This appeared to be due to most of the isocyanate becoming conjugated to proteins within the cell and thus not being extracted during the extraction procedure.A novel ethanol-saturated cellulose membrane blotting technique was developed for the extraction and ethanol-derivatisation of HDI from the surface of skin. However, not all of the HDI present on the membrane reacted with the ethanol. Increasing the amount of ethanol on the membrane did increase the amount of derivatised HDI monomer observed although this occurred at the expense of spatial information. The technique was also applied for analysis of the insecticide chlorpyrifos, for both skin surface sampling and permeation studies. From the images obtained, chlorpyrifos was observed to readily penetrate through the stratum corneum and reach a depth of 1.7mm. The highest amount was located in the dermis after the 1 hour exposure time.The dermal absorption of HDI was monitored after 1 hour exposure by mapping HDI monoamine penetration through the skin via indirect blotting and novel direct skin analysis methods. Similar profiles were observed from both methods. Penetration depths of 2.3 and 2.6 mm were observed for the direct skin and indirect blotting methods respectively. The highest level of HDI monoamine was located in the dermis

Application of imaging MALDI-MS to the determination of hazardous compounds in skin.

The overall aim of this work described in this thesis was to apply MALDI-MS and imaging MALDI-MS (MALDI-MSI) to the analysis of compounds of interest in occupational hygiene monitoring.Isocyanates are highly reactive compounds with a wide variety of industrial uses. MALDI-MS and MS/MS were used to investigate diisocyanate stability and reactivity. A monoisocyanate intermediate product was observed from the hydrolysis of both an aromatic and also an aliphatic diisocyanate. The stability of this product was assessed over a 14 day experimental period. Ethanol was used to derivatise hexamethylene diisocyanate (HDI) and the resulting urethane was observed to be stable over 14 days. This derivatisation method was incorporated into a surface swab technique for sampling of HDI from work surfaces.Industrial diisocyanates have been reported to penetrate through skin and to be excreted as diamine metabolites in urine. A LC-MS method for the determination of free toluene diamine (TDA) monomer formed by biotransformation of toluene diisocyanate (TDI) when applied to HaCaT cells was developed. In the two experiments performed, TDA was only observed at low levels after spiking with high concentrations of TDI. This appeared to be due to most of the isocyanate becoming conjugated to proteins within the cell and thus not being extracted during the extraction procedure.A novel ethanol-saturated cellulose membrane blotting technique was developed for the extraction and ethanol-derivatisation of HDI from the surface of skin. However, not all of the HDI present on the membrane reacted with the ethanol. Increasing the amount of ethanol on the membrane did increase the amount of derivatised HDI monomer observed although this occurred at the expense of spatial information. The technique was also applied for analysis of the insecticide chlorpyrifos, for both skin surface sampling and permeation studies. From the images obtained, chlorpyrifos was observed to readily penetrate through the stratum corneum and reach a depth of 1.7mm. The highest amount was located in the dermis after the 1 hour exposure time.The dermal absorption of HDI was monitored after 1 hour exposure by mapping HDI monoamine penetration through the skin via indirect blotting and novel direct skin analysis methods. Similar profiles were observed from both methods. Penetration depths of 2.3 and 2.6 mm were observed for the direct skin and indirect blotting methods respectively. The highest level of HDI monoamine was located in the dermis

Skin Tissue Models

Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio

Algorithmic assessment of cellular senescence in experimental and clinical specimens

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies—pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells—that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies

Advances on thirdhand smoke using targeted and untargeted approaches

El fum de tabac residual (thirdhand smoke en anglès, THS) és una via d'exposició a compostos tòxics de fum tabac poc estudiada fins ara. El THS es produeix per la deposició de parEcules i gasos en superFcies i pols, on es poden reemetre i/o reaccionar produint nous compostos tòxics, alguns d'ells carcinògens. Malgrat les creixents evidències, els riscos inherents a l'exposició a THS encara no s'han descrit completament. L'objecKu principal d'aquesta tesi és avançar en la caracterització química del THS i dels efectes per a la salut derivats d'aquesta exposició mitjançant l'aplicació de mètodes analíKcs dirigits i no dirigits. Aquesta tesi presenta el desenvolupament de un nou mètode analíKc per determinar simultàniament tòxics del tabac en pols domèsKca, mitjançant cromatografia líquida (UHPLC). En aquesta tesi, també s'ha desenvolupat un mètode d'anàlisi no dirigit basat en l'adquisició de mostres per UHPLC acoblada a espectrometria de masses d'alta resolució (HR-MS), amb l'aplicació d'estratègies avançades de processament de dades, la priorització estadísKca d’ions rellevants i una nova estratègia per a l'anotació de compostos. La combinació d'aquests dos mètodes va proporcionar per primera vegada l'anotació de dotzenes de tòxics relacionats amb la contaminació per THS adherits a la pols domèsKca. Pel que fa als efectes sobre la salut, presentem el primer estudi metabolòmic no dirigit en fetge de ratolins exposats a THS. L'aplicació de les tècniques UHPLC-HRMS i ressonància magnèKca nuclear (RMN) va permetre idenKficar dotzenes de metabòlits hepàKcs alterats, mentre que les imatges d'espectrometria de masses (MSI) van mostrar la distribució espacial diferencial de lípids en fetge induïda per THS. Aquests resultats confirmen els perills de l'exposició a THS i el paper clau de la introducció de noves estratègies metodològiques en la invesKgació en salut ambiental.El humo de tabaco residual (thirdhand smoke en inglés, THS) es una vía de exposición a compuestos tóxicos del humo del tabaco poco estudiada hasta la fecha. El THS se produce por la deposición de parBculas y gases en superficies y polvo, dónde se pueden reemiEr y/o reaccionar produciendo nuevos compuestos tóxicos, algunos de ellos carcinógenos. A pesar de las crecientes evidencias, los riesgos inherentes a la exposición a THS aún no se han descrito por completo. El objeEvo principal de esta tesis es avanzar en la caracterización química del THS y de los efectos para la salud derivados de esta exposición mediante la aplicación de métodos analíEcos dirigidos y no dirigidos. Esta tesis presenta el desarrollo de un nuevo método analíEco para determinar simultáneamente tóxicos del tabaco en polvo domésEco mediante cromatograMa líquida (UHPLC). En esta tesis, también se ha desarrollado un método de análisis no dirigido basado en la adquisición de muestras por UHPLC acoplada a espectrometría de masas de alta resolución (HR-MS), con la aplicación de estrategias avanzadas de procesamiento de datos, la priorización estadísEca de iones relevantes y una nueva estrategia para la anotación de compuestos. La combinación de estos dos métodos proporcionó por primera vez la anotación de docenas de tóxicos relacionados con la contaminación por THS adheridos al polvo domésEco.Respecto a los efectos sobre la salud, presentamos el primer estudio metabolómico no dirigido en hígado de ratones expuestos a THS. La aplicación de las técnicas UHPLC-HRMS y resonancia magnéEca nuclear (RMN) permiEó idenEficar docenas de metabolitos hepáEcos alterados, mientras que las imágenes de espectrometría de masas (MSI) mostraron la distribución espacial diferencial de lípidos en hígado inducida por THS. Estos resultados confirman los peligros de la exposición a THS y el papel clave de nuevos enfoques metodológicos en la invesEgación en salud ambiental.Thirdhand tobacco smoke (THS) is a novel and poorly understood pathway of tobacco exposure produced by the deposi=on and ageing of tobacco smoke par=cles and toxicants in surfaces and dust. This aged tobacco smoke could reemit into the air or react with other atmospheric chemicals to yield new toxicants, including carcinogens and becoming increasingly toxic with age. Although growing evidences of THS hazards, its chemical characteriza=on and the related health effects remain to be fully elucidated. Hence, this thesis aims to advance on the current knowledge on THS chemical characteriza=on and on the health effects derived from THS exposure by applying novel targeted and untargeted approaches. To advance on THS chemical characteriza=on, we have developed an efficient, quick and robust analy=cal method for simultaneously determining tobacco-specific compounds in household dust by ultra-highperformance liquid-chromatography coupled to tandem mass spectrometry (UHPLC-MSMS). We applied this target method in combina=on with untargeted strategies for a comprehensive characteriza=on of THS toxicants aNached to household dust. The developed untargeted workflow combines the sample acquisi=on by UHPLC coupled to high-resolu=on mass spectrometry (HR-MS) with the applica=on of advanced data processing strategies, the sta=s=cal priori=za=on of relevant features and a novel strategy for compound annota=on. The combina=on of these two approaches provided for the first =me the annota=on of dozens of toxicants related to THS contamina=on. To advance on the health effects, this thesis presents the first mul=plaQorm untargeted metabolomics study to unravel the molecular altera=ons of liver from mice exposed to THS. UHPLC-HRMS and nuclear magne=c resonance (NMR) revealed dozens of hepa=c metabolites altered in THS-exposed mice whilst mass spectrometry imaging (MSI) showed the differen=al spa=al distribu=on of lipids induced by THS. The results presented here confirm the hazards of THS exposure and the key role of combined untargeted and targeted methods in environmental health research

Proteomic analysis of cellular changes after treatment of human monocytic cell lines and primary dendritic cell models with contact allergens

Allergic contact dermatitis (ACD) is one of the most common immunotoxicological disorders of the skin. ACD is provoked by exogenous contact allergens that are mistakenly identified as a danger by the immune system and orchestrated by a T cell-mediated adaptive immune response. However, before a symptomatic response occurs (elicitation), a clinically silent sensitization phase precedes. The sensitization phase is characterized by complex interactions of different cell types with dendritic cells (DC) playing a central role. DCs may take up complexes of chemically-modified self-proteins, process and present them on their surface for T cell recognition. Subsequently, they undergo a maturation process and migrate to lymph nodes to initiate the priming and proliferation of allergen-specific T cells. The DCs are thus of crucial importance for the development of contact allergy. Despite this central role, the exact cellular signaling pathways leading to allergen-induced maturation of dendritic cells are not yet fully understood. As the current knowledge is mainly based on transcriptome data, state of the art mass spectrometry-based proteomics are applied in this thesis to complement and extend existing data to enhance the understanding of ACD pathomechanisms. In this work, it was shown for the first time that proteomic methods are suitable to distinguish activation of monocyte-derived dendritic cells (MoDC) triggered by the contact allergen NiSO4 from the bacterial activator lipopolysaccharide (LPS). NiSO4 caused not only a different MoDC phenotype but also the activation of divergent signaling pathways. Both treatments induced metabolic reprogramming towards aerobic glycolysis, which, however, manifested over a longer period in the case of NiSO4 and eventually activated hypoxia-like signaling pathways such as hypoxia-inducible factor (HIF) 1α upregulation. NiSO4 treatment further elicited a pronounced nuclear factor erythroid 2-related factor (Nrf) 2-dependent stress response in MoDCs. Most striking was the selective upregulation of cholesterol biosynthesis after treatment with NiSO4 which was absent upon stimulation with LPS. Concomitantly, the cells exhibited a significant decrease in cellular cholesterol levels. Thus, this signaling pathway was identified as a promising approach in the search for predictive biomarker signatures for sensitization with contact allergens. The second study was designed to verify the above-mentioned results in another cell model. Additional allergens were included to reveal similarities or differences in the activation of DCs by the allergens. THP-1 cells, which are standardly used for the prediction of contact allergens in vitro, were chosen as the cell model. The allergens NiSO4, p-dinitrochlorobenzene (DNCB), p-benzoquinone (BQ), and p-nitrobenzyl bromide (NBB) induced comparable proteomic changes and differed mainly in magnitude of induced proteomic changes. DNCB and NBB were the most potent activators of the cells. In direct comparison with MoDCs, THP-1 cells showed a much more pronounced Nrf2-mediated stress response. Based on these results, the Nrf2-mediated stress response should also be implemented into future in vitro DC test strategies for the prediction of contact allergens. Allergen-treated THP-1 cells developed a proinflammatory phenotype, which was supported by significant upregulation of the citrate cycle. Cholesterol biosynthesis was also significantly regulated in these cells - but in opposing direction to the findings from MoDCs. Overall, the comparison of MoDCs and THP-1 cells revealed relevant differences in the regulation of many proteins and signaling pathways between the cell models, but still resulted in the activation of similar superordinate systemic endpoints. THP-1 cells are therefore limited in their representation of MoDCs as primary DCs model in vitro. The further development of already validated in vitro methods in THP-1 cells is should thus focus on proteins that are differentially regulated by contact allergens in both cell types. Die allergische Kontaktdermatitis (ACD) ist eine der häufigsten immunotoxikologischen Erkrankungen der Haut. Hierbei wird das körperfremde Kontaktallergen vom Immunsystem fälschlicherweise als Gefahr identifiziert und eine T zellvermittelte adaptive Immunreaktion initiiert. Bevor eine gezielte Immunantwort in der Auslösephase erfolgen kann, geht eine asymptomatische Sensibilisierung voran. Die Sensibilisierungsphase ist ein komplexes Zusammenspiel verschiedener Immunzellen. Eine zentrale Rolle spielen hierbei die dendritischen Zellen (DC). Diese können Komplexe aus körpereigenen Proteinen mit den Kontaktallergenen erkennen, aufnehmen, prozessieren und auf ihrer Oberfläche den T Zellen präsentieren. Hierbei durchlaufen sie einen Reifungsprozess und wandern in Lymphknoten, um dort die Bildung und Proliferation allergen-spezifischer T Zellen einzuleiten. Die DCs sind somit von elementarer Bedeutung für die Ausprägung einer Kontaktallergie. Trotz dieser zentralen Rolle, sind die exakten zellulären Signalwege, die zu der allergeninduzierten Reifung der dendritischen Zellen führen, noch nicht vollständig verstanden. Da der derzeitige Wissensstand hauptsächlich auf Transkriptomdaten beruht, werden in dieser Dissertation moderne massenspektrometrische Proteomanalysen durchgeführt, um die vorhandenen Daten zu ergänzen sowie zu erweitern und so die zugrundeliegenden Pathomechanismen von ACD besser zu verstehen. Im Rahmen dieser Arbeit konnte erstmals gezeigt werden, dass proteomische Methoden geeignet sind, die Aktivierung von MoDCs durch das Kontaktallergen NiSO4 von dem bakteriellen DC Aktivator Lipopolysaccharid (LPS) zu unterscheiden. NiSO4 induzierte nicht nur einen anderen Phänotyp, sondern auch die Aktivierung divergenter Signalwege in den Zellen. Beide Behandlungen induzierten eine metabolische Reprogrammierung hin zur aeroben Glykolyse, welche sich jedoch im Fall von NiSO4 über einen längeren Zeitraum manifestierte und schließlich Hypoxie-ähnliche Signalwege wie hypoxia-inducible factor (HIF) 1α aktivierte. Die NiSO4-Behandlung löste weiterhin eine ausgeprägte nuclear factor erythroid 2-related factor (Nrf) 2-abhängige Stressantwort in MoDCs aus. Besonders prominent war zudem die selektive Hochregulation der Cholesterolbiosynthese nach Behandlung mit NiSO4. Dies ging mit einer signifikanten Verminderung der zellulären Cholesterollevel einher. Dieser Signalweg stellt somit einen vielversprechenden Ansatz auf der Suche nach prädiktiven Biomarkersignaturen für die Sensibilisierung mit Kontaktallergenen dar. In einer zweiten Studie sollten die Ergebnisse der ersten Studie in einem anderen Zellmodell verifiziert und weitere Allergene untersucht werden, um Gemeinsamkeiten oder Unterschiede in der Aktivierung von DCs durch die Allergene aufzuzeigen. Als Zellmodell wurden THP-1 Zellen gewählt, welche standardmäßig für die Vorhersage von Kontaktallergenen in vitro genutzt werden. Die Allergene NiSO4, p-Dinitrochlorbenzol (DNCB), p-Benzoquinon (BQ) und p-Nitrobenzylbromid (NBB) induzierten vergleichbare proteomische Veränderungen und unterschieden sich hauptsächlich hinsichtlich ihrer Potenz. DNCB und NBB waren die potentesten Aktivatoren der Zellen. Im direkten Vergleich zu den MoDCs zeigten THP-1 Zellen eine deutlich ausgeprägtere Nrf2-vermittelte Stressantwort. Basierend auf diesen Ergebnissen sollte die Nrf2-vermittelte Stressantwort künftig auch in in vitro Teststrategien für die Vorhersage von Kontaktallergenen in DCs implementiert werden. Allergen-behandelte THP-1 Zellen entwickelten einen proinflammatorischen Phänotyp, welcher durch signifikante Hochregulation des Citratzyklus gestützt wurde. Auch in diesen Zellen war die Cholesterolbiosynthese signifikant reguliert – jedoch gegensätzlich zu den Befunden in den MoDCs. Insgesamt ergaben sich im Vergleich von MoDCs und THP-1 Zellen gravierende Unterschiede die Regulierung vieler Proteine und Signalwege betreffend, dennoch resultierte dies in der Aktivierung übergeordneter systemischer Endpunkte. THP-1 Zellen eignen sich folglich nur begrenzt als Modell für die Aktivierung von primären DCs in vitro. Die Weiterentwicklung von bereits validierten in vitro Methoden in THP-1 Zellen sollte sich somit auf Proteine stützen, die in beiden Zellentypen gleichsam durch Kontaktallergene exprimiert werden