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Mast cells reside on and near the cerebral vasculature, the predominant site of pneumococcal entry into the central nervous system (CNS). Although mast cells have been reported to be crucial in protecting from systemic bacterial infections, their role in bacterial infections of the CNS remained elusive. Here, we assessed the role of mast cells in pneumococcal infection in vitro and in vivo. In introductory experiments using mouse bone marrow-derived mast cells (BMMC), we found that (i) BMMC degranulate and release selected cytokines upon exposure to Streptococcus pneumoniae, (ii) the response of BMMC varies between different pneumococcal serotypes and (iii) is dependent on pneumolysin. Intriguingly though, apart from a slight enhancement of cerebrospinal fluid (CSF) pleocytosis, neither two different mast cell-deficient Kit mutant mouse strains (WBB6F1-KitW/Wv and C57BL/6 KitW-sh/W-sh mice) nor pharmacologic mast cell stabilization with cromoglycate had any significant impact on the disease phenotype of experimental pneumococcal meningitis. The incomplete reversal of the enhanced CSF pleocytosis by local mast cell engraftment suggests that this phenomenon is caused by other c-Kit mutation-related mechanisms than mast cell deficiency. In conclusion, our study suggests that mast cells can be activated by S. pneumoniae in vitro. However, mast cells do not play a significant role as sentinels of pneumococcal CSF invasion and initiators of innate immunity in vivo.
The autism susceptibility locus on human chromosome 7q32 contains the maternally imprinted MEST and the non-imprinted COPG2 and TSGA14 genes. Autism is a disorder of the ‘social brain’ that has been proposed to be due to an overbalance of paternally expressed genes. To study regulation of the 7q32 locus during anthropoid primate evolution, we analyzed the methylation and expression patterns of MEST, COPG2, and TSGA14 in human, chimpanzee, Old World monkey (baboon and rhesus macaque), and New World monkey (marmoset) cortices. In all human and anthropoid primate cortices, the MEST promoter was hemimethylated, as expected for a differentially methylated imprinting control region, whereas the COPG2 and TSGA14 promoters were completely demethylated, typical for transcriptionally active non-imprinted genes. The MEST gene also showed comparable mRNA expression levels in all analyzed species. In contrast, COPG2 expression was downregulated in the human cortex compared to chimpanzee, Old and New World monkeys. TSGA14 either showed no differential regulation in the human brain compared to chimpanzee and marmoset or a slight upregulation compared to baboon. The human-specific downregulation supports a role for COPG2 in the development of a ‘social brain’. Promoter methylation patterns appear to be more stable during evolution than gene expression patterns, suggesting that other mechanisms may be more important for inter-primate differences in gene expression.
Differences in Mouse Hepatic Thyroid Hormone Transporter Expression with Age and Hyperthyroidism
(2015)
Background: Clinical features of thyroid dysfunction vary with age, and an oligosymptomatic presentation of hyperthyroidism is frequently observed in the elderly. This suggests age modulation of thyroid hormone (TH) action, which may occur, for example, by alterations in TH production, metabolism and/or TH action in target organs. Objectives: In this paper, we address possible changes in TH transporter expression in liver tissues as a mechanism of age-dependent variation in TH action. Methods: Chronic hyperthyroidism was induced in 4- and 20-month-old C57BL6/NTac male mice (n = 8-10) by intraperitoneal injections of 1 µg/g body weight <smlcap>L</smlcap>-thyroxine (T<sub>4</sub>) every 48 h over 7 weeks. Control animals were injected with PBS. Total RNA was isolated from liver samples for analysis of the TH transporter and TH-responsive gene expression. TH concentrations were determined in mice sera. Results: Baseline serum free T<sub>4</sub> (fT<sub>4</sub>) concentrations were significantly higher in euthyroid young compared to old mice. T<sub>4</sub> treatment increased total T<sub>4</sub>, fT<sub>4</sub> and free triiodothyronine to comparable concentrations in young and old mice. In the euthyroid state, TH transporter expression was significantly higher in old than in young mice, except for Mct8 and Oatp1a1 expression levels. Hyperthyroidism resulted in upregulation of Mct10, Lat1 and Lat2 in liver tissue, while Oatp1a1, Oatp1b2 and Oatp1a4 expression was downregulated. This effect was preserved in old animals. Conclusion: Here, we show age-dependent differences in TH transporter mRNA expression in the euthyroid and hyperthyroid state of mice focusing on the liver as a classical TH target organ.
Die vorliegende Arbeit adressiert die Nutzbarkeit des humanen Speichelproteoms als diagnostisches Instrument im Kontext einer oralen Mukositis bei Kopf- und Halskarzinoms. Als häufigste Nebenwirkung einer Radio(chemo)therapie kann die Mukositis therapielimitierend sein und hat für betroffene Patienten meist eine Einschränkung ihrer Lebensqualität zur Folge. Trotz der guten Verfügbarkeit von Speichel existieren wenige Studien, welche zeigen, dass das Speichelproteom für die Diagnostik einer Krankheit oder zur Therapieentscheidung nutzbar ist. Das hat unter anderem seinen Grund in der Komplexität der massenspektrometrischen Methode. Die erste Veröffentlichung (Golatowski et al. 2013) erarbeitete deshalb einen Standard in der Probengewinnung von Speichel. Als Ergebnis steht die Empfehlung zur Nutzung eines Paraffin-Kaugummis, aufgrund des hohen Speichelvolumens und der guten Vergleichbarkeit mit der nichtstimulierten Salivation beim identifizierten Proteom. In einer zweiten Veröffentlichung (Jehmlich & Golatowski et al. 2014) wurden C18 Mikrosäulen verschiedener Hersteller bezüglich ihres Einflusses auf die Proteinidentifizierung verglichen. Die Säulen sind notwendig für die Entsalzung und Aufreinigung eines Peptidgemisches. Mit allen verwendeten Säulen konnten ähnliche Ergebnisse erzielt werden, wobei die ZipTip® µC18 sowie C18 Systeme der OASIS® HLB μElution 96er Well Platte und TopTip® C18 Pipettenspitzen leicht überlegen sind. In der letzten Arbeit (Jehmlich et al. 2015) wurden die gewonnenen Erkenntnisse genutzt, um die Speichelproben von Patienten mit Kopf- und Halskarzinom zu untersuchen. Insgesamt zeigten wir die Möglichkeit, alterierte Proteine zwischen zwei Patientengruppen massenspektrometrisch zu detektieren. Mit den gefundenen Daten konnte demonstrieren werden, dass massenspektrometrische Techniken geeignet sind, um schon vor Behandlungsbeginn Patienten zu identifizieren, die für die Entwicklung einer oralen Mukositis prädisponiert sind. Es ist hierbei die Proteinklasse der Metalloproteinasen hervorzuheben, da diese für einen therapeutischen Ansatz gegen Mukositis interessant sind. In Zukunft werden jedoch größere und voraussichtlich multizentrische Studien erforderlich sein, um ausreichend große Patientenkohorten zusammenzustellen und die Klassifikation speziell für Patienten ohne Mukositisrisiko sensitiver zu gestalten.
Humanity is plagued by many diseases. Beside environmental influences, many --- if not all --- diseases are also subject to genetic predisposition and then display molecular alterations such as proteomic or metabolic aberrations. The elucidation of the molecular principles underlying human diseases is one of the prime goals of biomedical research. To this end, there has been an advent of large-scale omics profiling studies. While the field of molecular biology has experienced tremendous development, data analysis remains a bottleneck. In the context of this thesis, we developed a number of analysis strategies for different types of omics data resulting from different experimental settings. These include approaches for associations studies for plasma miRNAs and time-resolved plasma omics data. Furthermore, we devised analyses of different RNA-Seq transcriptome profiling studies coping with problems such as lack of replicates or multifactorial experimental design. We also designed machine learning frameworks for the identification of discriminatory biomolecular signatures analysing case-control or time-to-event data. All of the strategies mentioned above were developed and applied in the contexts of multi-disciplinary endeavours. They aided in the identification of plasma miRNAs associated with age, sex, and BMI as well as plasma miRNAs bearing potential as diagnostic biomarkers for non-alcoholic fatty liver disease (NAFLD). This thesis significantly contributed to a study demonstrating the utility of plasma miRNAs as prognostic biomarkers for major cardiovascular events such as ST-elevation myocardial infarction. Our approaches for analysing RNA-Seq data aided in the characterisation of murine models for Alzheimers disease and the transcriptional response of human gingiva fibroblasts to ionizing radiation exposure. Furthermore, the developed approaches were applied for studying a human model for thyrotoxicosis and for the successful identification of a multi-omics plasma biomarker signature of thyroid status. We are only beginning to understand the molecular principles underlying human diseases. The approaches and results presented in this thesis will contribute to improved understanding of biomolecular processes involved in common diseases such as Alzheimers disease, NAFLD, and cardiovascular diseases.
Transfusion-related acute lung injury (TRALI) is an adverse transfusion reaction and the major cause of transfusion-related mortality. The syndrome occurs within six hours after transfusion and is characterized by acute respiratory distress and the occurrence of a non-cardiogenic, bilateral lung edema. TRALI is almost entirely induced by leukocyte-reactive substances which are present in the blood product and get transferred to the recipient during transfusion. The majority of cases (~80%) is caused by leukocyte-reactive immunoglobulins and is accordingly classified as immune-mediated TRALI. The responsible antibodies are generated via alloimmunization and are directed against human leukocyte antigens of class I and II or human neutrophil alloantigens (HNA). Within the HNA class, HNA-3a antibodies have an exceptional clinical relevance as they are most frequently involved in severe and fatal TRALI cases. The high mortality was associated with their characteristic ability to induce a strong neutrophil aggregation response. The described clinical relevance of HNA-3a antibody-mediated TRALI motivates the screening for new strategies for preventive or acute pharmacologic intervention. Knowledge of the molecular pathomechanisms is a crucial prerequisite and thus, respective investigations are required. In order to achieve this goal, HNA-3a antibody-induced cytotoxicity and aggregation were assessed on the molecular level by usage of flow cytometry, the granulocyte agglutination test and by phosphoproteome analysis. The current study provides insight into molecular processes during HNA-3a antibody-induced neutrophil responses and is the first to assess neutrophils using global, gel-free phosphoproteome analyses. Accordingly, it is the first to provide neutrophil phosphoproteome data in the context of TRALI. Gel-free phosphoproteome analyses of primary neutrophils required the highly selective and sensitive phosphopeptide enrichment from stable and sufficiently large protein extracts. However, an appropriate workflow did not exist and was hence developed by sequential protocol optimization steps. The developed workflow was finally proven suitable for comparative gel-free phosphoproteomics when detecting the formyl-methionyl-leucyl-phenylalanine-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in a proof-of-principle experiment. The following single parameter analyses were conducted to investigate neutrophils for their responses to HNA-3a antibodies in absence and presence of proinflammatory priming conditions. Results revealed that the direct stimulation of neutrophils with HNA-3a antibodies will likely not cause the induction of cytotoxic effector functions. In contrast, neutrophils react predominantly by aggregation, a process which is potentially mediated by integrins and causes a secondary, subthreshold activation of solely ERK2. Accordingly, only the neutrophil aggregation response could also be enhanced by an appropriate priming. Taken together, the single parameter analyses proved neutrophil aggregation as the main pathomechanism in HNA-3a antibody-mediated TRALI and thus, the underlying signaling pathways were investigated by global, gel-free phosphoproteomics. The following phosphoproteome analyses indicated the induction of a biphasic signaling during 30 minutes of HNA-3a antibody treatment and signaling pathways of Rho family GTPases could be associated with the first and the second phase. Additionally, the involvement of ERK signaling was indicated in the second phase and this result corroborated thus the data of the previous single parameter analyses. The comprehensive analysis of the identified signaling pathways revealed Rho, Rac and Cdc42 as central regulators and the specific inhibition of Rho in the following validating experiments led very intriguingly to a significant enhancement of HNA-3a antibody-mediated neutrophil aggregation. Hence, this result indicated a potential inhibitory effect of HNA-3a antibodies on Rho activity. Therefore, Rho inhibition was suggested to occur in parallel to an adhesion-inducing signaling pathway and might hence be involved in the stabilization of neutrophil aggregates in HNA-3a antibody-induced TRALI. The results from this doctoral thesis contributed to the generation of a new pathogenesis model for HNA-3a antibody-mediated TRALI. In this model, neutrophils respond to direct HNA-3a antibody exposure predominantly by homotypic aggregation. These potentially very stable and primed aggregates accumulate in the lung and are susceptible to parallel, proinflammatory stimulation. Subsequently, this cascade leads to full neutrophil activation and finally to TRALI induction.
Staphylococcus aureus (S. aureus) is the leading cause of serious diseases in human both from hospital and community associated infections. Some clinical manifestations of S. aureus infections are infective endocarditis (IE), osteoarticular infections, skin and soft tissue, pleuropulmonary, and device-related infections. In Germany, S. aureus is the second most common cause of hospital-acquired (HA) infections. About 16.7% of these nosocomial infections are caused by HA-MRSA clinical isolates. It has been a huge threat for the clinicians/scientists to control the emergence of such infections caused by S. aureus. S. aureus exhibits increasing virulence and resistance to various antibiotics, complicating prevention and treatment of infections. Eventually, active and passive vaccines might be the alternative strategy to deal with S. aureus related diseases. An effective S. aureus vaccine would provide great potential security and many societal benefits. However, so far vaccine trials have failed often due to limited number of available antigen candidates (monovalent/single antigen) in the clinical trials. Efforts to develop not only S. aureus vaccine but also prognosis or diagnosis tools are challenging tasks. That was the motivation point for the current thesis to identify potential antigen candidates for the aid of vaccine development using immunoproteomics approaches. From the earlier studies, passive immunisation with CP5, CP8, PNAG, ClfA, SdrG, alpha-hemolysin and active immunisation with IsdB, SEB, ClfA, CP5, CP8 were examined during preclinical trials and found to be the best examples for potential vaccine candidates. The antibody responses against S. aureus infections are heterogenous, still it is possible to identify the antibody signatures to a number of corresponding S. aureus antigens, whose abundance and presence could correlate to the disease state and may predict treatment outcome. To support this hypothesis, goals were set to develop and validate serological assay by indirect detection using suspension array technology (SAT). During the study, an antigen library of 140 recombinant S. aureus antigens was generated. Further serological assay were developed and validated to monitor the insights of antibody mediated humoral responses during S. aureus infection from various episodes of S. aureus infection. As an outcome, potential immunogenic antigen candidates were identified which may be used as candidates in active/passive vaccination and to stratify the patient. In total, three studies were carried out using serum and plasma samples from S. aureus nasal colonised healthy individuals (carriers and non-carriers) and bacteraemia patients (control, complicated and uncomplicated sepsis). Bead-based assays were performed and subsequent statistical analyses were done to identify immunogenic antigens that might discriminate between the different clinical status and outcome. Screening of healthy individuals (study-1) have shown significantly higher IgG responses against 14 antigens in S. aureus nasal carriers compared to non-carriers. Furthermore, the clonal complex 30 group of healthy carriers has shown significantly higher IgG responses against toxic shock syndrome toxin-1 (Tsst1) in comparison to non-clonal complex 30 healthy carriers. Study-2 have shown extensively higher IgG responses against 67 antigens in control samples compared to sepsis patients. 50% of the antigens eliciting different immune responses belonged to the extracellular components of S. aureus. The IgG responses against MSCRAMM proteins such as FnbA, FnbB, Efb-1 have been shown to be significantly higher in complicated sepsis. Study-3 have shown notably higher IgG responses against 8 antigens (Plc, SspB, IsaA, SEM, GlpQ, HlgC, SACOL0444, SACOL0985) at baseline in uncomplicated sepsis patients compared to patients subsequently developing complicated sepsis. In summary, the group of immunogenic antigens that have been identified in these studies using immunoproteomics approach could be a starting point for the development of S. aureus vaccines. Moreover, the suspension array technology approach facilitated the identification of new S. aureus antigen candidates in addition to earlier reports. The current results of this study support the hypothesis that it is possible to identify a serological response to potential S. aureus antigens that correlate to progression of S. aureus infections.
Dilated cardiomyopathy (DCM) is a myocardial disorder characterised by ventricular dilation with reduced left ventricular ejection fraction (LVEF). Immunoadsorption (IA) followed by immunoglobulin (IgG) substitution (IA/IgG) has been shown to be a promising therapeutic intervention to recover myocardial functions in DCM patients. The beneficial effects of IA/IgG therapy are associated with increased LVEF, decreased left ventricular inner diameter at diastole (LVIDd) and reduced myocardial inflammation. Despite knowing the cardiac benefits of IA/IgG, the precise molecular mechanism induced by therapy is still elusive. Additionally, only ≈60 % DCM patients treated with IA/IgG demonstrated improved heart function. Moreover, the reasons for this differential outcome among DCM patients after treatment have not been clearly understood. In this study, efforts were made to uncover the therapy induced proteomic changes in the heart of responders (relative change in LVEF ≤ 20%, LVEF < 5% absolute value) and non-responders using a global proteomic approach. Apart from it, proteomic profiling of endomyocardial biopsies and plasma was performed to find protein biomarker candidates which might be useful to distinguish responder and non-responder DCM patients before immunoadsorption therapy and support a selective and individualized treatment. To reveal therapy induced myocardial proteomic changes, endomyocardial biopsies of DCM patients before and after therapy were compared. LVEF increased (32 ± 8 to 45±7, p<0.002) and LVIDd decreased (66 ± 6 to 60±6, p<0.040) after therapy in responders, whereas non-responders did not show any significant changes in these clinical parameters. To address the changes in the myocardial proteome induced by therapy, a label-free proteomic approach was applied. The most prominent proteomic differences between both subgroups were observed in cytoskeletal, fibrosis, and extracellular matrix proteins. Therapy linked benefit in responders seems to be highly associated with the lower abundance of fibrotic and extracellular matrix proteins which seems to reflect a lower activity of transforming growth factor-β signaling. To elucidate proteomic differences between responders and non-responders at baseline, endomyocardial biopsies and plasma proteome profiling were performed. Responder and non-responder DCM patients did not show any significant differences in the clinical parameters (LVEF, LVIDd, age, inflammation, etc.) before IA/IgG therapy except for disease duration that was in tendency higher among non-responders. Proteomics profiling of endomyocardial biopsies revealed 54 differentially abundant proteins between responders and non-responders. Among those proteins, Protein S100-A8 and kininogen-1 was found higher whereas perilipin-4 was found lower abundant in responders. Plasma profiling of these subgroups revealed five proteins (S100-A8, S100-A9, C-Reactive protein, lipopolysaccharide-binding protein, and cysteine-rich secretory protein) displaying strong discriminative power between responders and non-responders. Higher abundance of Protein S100-A8 was observed in myocardium as well as in plasma among responders. Protein S100-A8 might be a potential candidate to distinguish responders and non-responders at baseline, and its potential utility at clinical levels must be evaluated. The last objective of the thesis was to establish a workflow for the relative quantitation of phosphopeptides for samples generally obtained in small amounts like myocardial biopsies. To address this question, optimization was performed with HL-1 cardiomyocytes using a PolyMAC phosphopeptide enrichment kit and the effect of TGF-β1 on the phosphoproteome was evaluated as a proof-of-principle study. Using only 200µg protein of each sample up to 2000 phosphopeptides with an efficiency of >90 percent could be covered. In total, upon TGF-β1 incubation alterations of 214, 92, and 53 phosphopeptides were observed after 1, 6 and 24 hours, respectively. Differentially altered phosphopeptides belonged to many signaling pathways including the ubiquitin-proteasome pathway, cytoskeletal regulation by Rho GTPase, calcium signaling, and TGF-β signaling. Thus, in this study a workflow for relative quantitation of phosphopeptides was established that may be later applied to precious biopsy samples. Along with this, TGF- β1 induced phosphoproteome was analysed in HL-1 cardiomyocytes.
Die Transfusionsassoziierte Akute Lungeninsuffizienz (TRALI) ist die häufigste tödliche Nebenwirkung der Transfusion von Blutprodukten und wird oft durch mittransfundierte leukozytenreaktive Antikörper (AK) induziert. AK gegen das Humane Neutrophilenantigen (HNA)-3a verursachen häufig schwere Fälle der TRALI. HNA-3a ist auf dem Großteil der Blutzelltypen exprimiert und entsteht durch einen Einzelnukleotidpolymorphismus im Gen des „choline transporter-like protein 2“ (CTL2), welcher zur Substitution der Aminosäureposition 154 in der Sequenz des Proteins führt. Klinische Beobachtungen und zahlreiche Studien legen nahe, dass TRALI-induzierende AK die Akkumulation und Aktivierung von Granulozyten im Lungenkapillarbett verursachen. Durch den Zusammenbruch der Kapillarbarriere kommt es in der Folge zu einem Lungenödem. Die Entwicklung eines Hochdurchsatzverfahrens zur Detektion von HNA-3a-AK in Blutprodukten und die Identifizierung therapierelevanter Schaltstellen, im Pathomechanismus der HNA-3a-AK-induzierten TRALI, waren daher Hauptschwerpunkte dieser Studie. In diesem Zusammenhang wurde ein Nachweissystem für HNA-3a-AK auf der Grundlage von CTL2-Fragmenten etabliert. Ein Screening zahlreicher Anti-HNA-3a-Plasmen ergab, dass mit CTL2-Peptiden in Festphasentests jedoch nur etwa 50% aller HNA-3a-AK nachgewiesen werden können. Weitere Untersuchungen ergaben, dass die Detektion aller HNA-3a-AK nur mit zellbasierten Methoden möglich ist, bei denen CTL2 in seiner natürlichen Konformation vorliegt. Diese Studie leistet damit einen wichtigen Beitrag zur Charakterisierung des konformationssensitiven Epitops der HNA-3a-AK und identifiziert wichtige Grundvoraussetzungen für Methoden zum Nachweis dieser AK. Ein weiterer Schwerpunkt dieser Arbeit war die Untersuchung der Interaktion zwischen HNA-3a-AK und Granulozyten. Dieser experimentelle Ansatz gründet auf histopathologischen Lungenuntersuchungen verstorbener TRALI-Patienten, die eine massive Akkumulation und Aggregation von Granulozyten in den Kapillaren aufzeigen. Aus diesem Grund erfolgte die Untersuchung dieser Interaktion zunächst hinsichtlich verschiedener Parameter, die zu einer gesteigerten Sequestrierung von Granulozyten in der Lunge führen können. Hierzu gehören die Granulozytenaggregation (GA), die Versteifung von Zellen und die Zelladhäsion. Die Modifikation des Standard-Granulozytenaggregationstests ergab, dass die HNA-2- und HNA-3a-AK-induzierte GA aktive Prozesse sind, welche unabhängig von Plasmafaktoren stattfinden, jedoch von der Aktivität einer bislang noch nicht identifizierten Serinprotease abhängig sind. Hierbei wurden potente Aggregationsinhibitoren identifiziert welche auch als potenzielle Therapeutika zur Behandlung von TRALI in Frage kommen. Mit Hilfe desselben Testsystems konnte ermittelt werden, dass voraktivierte Granulozyten eine erhöhte Aggregationsneigung aufweisen. Im Zusammenhang mit dem Schwellenwertmodell der TRALI liefert dieses Ergebnis eine mögliche Erklärung, warum Patienten mit schweren Vorerkrankungen häufiger an TRALI erkranken. Neben der GA wurden in dieser Studie zwei weitere Eigenschaften identifiziert, die eine Akkumulation der Zellen in den engen Lungenkapillaren erklären: deren Elastizität und Adhäsivität. Mithilfe der Rasterkraftmikroskopie wurde nachgewiesen, dass HNA-3a-AK eine Versteifung von Granulozyten induzieren. Ebenso wurde gezeigt, dass HNA-3a-AK das Integrin Mac-1 (CD11b/CD18) auf der Granulozytenoberfläche aktivieren, was zu einer verstärkten Adhäsion der Zellen an Fibrinogen führt. Steifere und adhäsivere Granulozyten akkumulieren möglicherweise vermehrt in den Lungenkapillaren. Eine weitere wichtige Fragestellung dieser Studie war, ob HNA-3a-AK eine direkte Aktivierung von Granulozyten auslösen. Die Untersuchungen ergaben, dass die Aktivierung von CD11b, weder mit der Erhöhung der CD11b-Expression, bzw. mit der proteolytischen Abspaltung von L-Selektin, noch mit einer Sauerstoffradikalproduktion einhergeht. Dieses ungewöhnliche Muster an Veränderungen deutet nicht auf eine Aktivierung zytotoxischer Antworten hin. Seit kurzem ist bekannt, dass HNA-3a-AK Kapillarendothelzellen auch auf direktem Wege aktivieren und dass selbst in Granulozyten-depletierten Mäusen schwache TRALI-Symptome auftreten. Demzufolge ergibt sich ein neues Modell der HNA-3a-AK-induzierten TRALI: Nach Transfusion von HNA-3a-AK bleiben aggregierte, steifere und adhäsivere Granulozyten in den engen Lungenkapillaren stecken und interagieren dort mit dem aktivierten Gefäßendothel. Durch diese Interaktion kommt es vermutlich zur Aktivierung der Granulozyten und zu einer starken Inflammationsreaktion, welche eine weitere Zerstörung der Lungenkapillaren und schließlich ein schweres Lungenödem zur Folge hat. Die besondere Schwere der HNA-3a-AK-induzierter TRALI entsteht also vermutlich nicht aufgrund einer direkten und starken Granulozytenaktivierung, sondern vielmehr durch die gleichzeitige Beeinflussung verschiedener epitoptragender Zelltypen.
Heart Failure is currently the most common cardiac disorder and a major public health concern worldwide. The adult mammalian heart harbors a subpopulation of cardiac progenitor cells (CPC) that are capable of improving cardiac function. The scope of this study was to delineate the molecular phenotype of a subpopulation of CPCs characterized by the expression of the stem cells antigen-1 surface marker (Sca-1+) and to further identify molecular alterations occurring under heart failure conditions. In order to understand the underlying cellular mechanisms an integrated approach of proteomics and transcriptomics-based techniques were employed. The first step towards achieving this goal was to unravel the native Sca-1+ cell characteristics of freshly isolated progenitor cells derived from healthy adult murine hearts. The proteome map of Sca-1 cells was established using a gel-based mass-spectrometry (gel LC-MS/MS) approach. For better interpretation, a comparison with the protein profiles of cardiomyocytes and Sca-1- cells obtained under similar experimental conditions was performed. All three cell-types were morphologically different in size and structure, which was also evident from their protein expression profiles. We observed that Sca-1+ cells lack endothelial-like and cardiac contractile phenotypes, unlike Sca-1- cells and cardiomyocytes, respectively. Functional assessment of both protein and gene expression profiles revealed a possible role of Sca-1+ cells in cell adhesion, migration, and proliferation. CPC remain in a dormant state under physiological condition unless challenged by myocardial injury. Previous studies revealed that resident Sca-1+ cells home to the injured myocardium but not to the healthy heart and further differentiate into functional cardiomyocytes. We investigated the molecular background of this behavior of adult Sca-1+ cells under heart failure condition which might provide a better insight into their cardiogenic potential in a pathological milieu. The double transgenic α-myosin heavy chain (MHC)-cyclin T1/Gαq overexpressing mouse was chosen as a model for heart failure. Using the comparative gene expression profiling we could detect the differential regulation of 197 genes with at least a 2-fold difference. Among these BDNF mRNA levels were 5-fold higher in the Sca-1+ cells derived from transgenic mice (Cyc+) in comparison to that of wild-type controls (Wt+). This difference was also observed at protein level. The substantially higher expression of BDNF during heart failure prompted us to investigate its regulatory effect on Sca1+ cells. In this current study we were able to show that small amounts of exogenous BDNF stimulated the migratory potential of Cyc+ cells. This effect was not seen in treated Wt+ cells. Furthermore, pulsed SILAC was employed to monitor BDNF mediated changes following treatment. After BDNF treatment, 58 proteins were differentially regulated of which proteins related to cell proliferation were reduced in level in Cyc+ cells while they displayed increased levels in Wt+ cells. Findings from bromodeoxyuridine (BrdU) assays and immunoblotting indicated that BDNF might initiate a differentiation program by repressing cell proliferation in Cyc+ cells. Taken together, it could be shown that the BDNF effect on protein synthesis of Cyc+ and Wt+ cells varied considerably, suggesting an improvement of the cardiogenic potential of Sca-1+ cells under pathological conditions. Aldosterone levels are known to be elevated during heart failure. In this part of study it was hypothesized that endocrine factors associated with heart failure might influence the migration of CPC, thereby possibly restoring the cardiac function of diseased hearts. It could be shown that high concentrations of aldosterone, similar to those found in the plasma of heart failure patients, induced the migration of Sca-1+ cells by up to 60% when compared to control, while physiological levels had no significant influence. In addition, it could be demonstrated that the aldosterone stimulus led to the activation of the mineralocorticoid receptor (MR) expressed on Sca1+ cells, which in turn facilitated migration. This was supported by application of MR antagonist eplerenone, which significantly reduced the aldosterone-induced increase in cell migration while a glucocorticoid antagonist exhibited no inhibitory effect. Hence, the results support the potential role of aldosterone in the mobilization of CPC. It is currently believed that the beneficial effects of cell-based therapies on cardiac repair are imparted to a large degree via paracrine mechanisms. We therefore focused on understanding the influence of pathophysiological levels of aldosterone on the extracellular environment of Sca-1+ cells. MS-based secretome profiling of cells treated for 24h with aldosterone treatment revealed higher levels of proteins associated with extracellular matrix remodeling and IGF signaling. Additionally, galectin-1 and gelsolin were significantly increased in level under pathological conditions indicating a possible paracrine tissue repair of Sca-1+ cells. To conclude, the global proteome and transcriptome profiles generated here revealed the molecular phenotype of Sca-1+ cells which may be used for future reference. The comparative microarray study provided deeper insight into the endogenous changes in mRNA expression during heart failure and delineated the cardiogenic characteristics of Sca-1+ cells. Moreover, the data presented here shed new light on the potential role of BDNF in regulating the mobilization and proliferation of CPCs. Our study on the influence of aldosterone on the migration and the extracellular proteome of CPCs provided new insights on the beneficial effects of this mineralocorticoid on cardiac cells.
