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Ontamalimab is a therapeutic candidate antibody targeting MAdCAM-1, intended for the treatment of inflammatory bowel diseases (IBD), of which the main entities are ulcerative colitis and Crohn’s disease. During such inflammation of the gut, T cells home to the affected tissue via the interaction of integrin α4β7 on the T cell surface with MAdCAM-1 on the surface of endothelial cells. Blockade of this interaction using the anti-α4β7 integrin antibody vedolizumab has been successfully used for the treatment of IBD since 2014. Nevertheless, not all patients benefit from this treatment. Therefore, the goal of this study was to investigate the mechanism of action of ontamalimab and to directly compare it with vedolizumab. Starting with general research about MAdCAM-1 itself, this work shows that IBD patients with acute inflammation express higher levels of MAdCAM-1 and that only one transcript variant is predominantly expressed. Several cytokines, transcription factors and adhesion molecules also showed altered expression in IBD patients compared to controls. While short chain fatty acids might reduce the expression of MAdCAM-1, TNFα was shown to induce it. TNFα was further associated with shedding of MAdCAM-1, which supports previous suggestions that soluble (s-)MAdCAM-1 may be a biomarker for intestinal inflammation. Ontamalimab caused the internalization of MAdCAM-1, thereby not only blocking its interaction to ligands, but also entirely removing it from the cell surface. Moreover, ontamalimab significantly inhibited the L-selectin/MAdCAM-1-mediated rolling of several immune cell subsets, including T cells, granulocytes and monocytes, while firm adhesion of several T cell subsets was equally blocked by ontamalimab and vedolizumab. Homing of T cells into the gut of mice with dextran sodium sulfate-(DSS-) induced colitis was significantly reduced by an anti-MAdCAM-1 treatment with MECA367 and to a lesser, not significant extent also by anti-α4β7 integrin (DATK32). After demonstrating that the two antibody treatments affect the T-cell homing process differently, further effects of an anti-MAdCAM-1 treatment were investigated in experimental colitis models, also with regard to innate immune cells, in comparison to an anti-α4β7-integrin treatment. In a T cell transfer colitis model, DATK32 and MECA367 treatment led to significantly reduced histological scores and diminished CD4+ T cell abundance in the colon. However, weight loss and abundance of innate immune cells remained unaffected by either treatment. Similar results were obtained in DSS-colitis experiments, that are not primarily T cell-dependent. Moreover, the wound healing process in mice without intestinal inflammation was not affected by DATK32 or MECA367 administration.
In conclusion, these data show that MAdCAM-1 expression and regulation play an important role for the pathogenesis of IBD. Therapeutic blockade of MAdCAM-1 with ontamalimab has a distinct mechanism that is different from α4β7 integrin blockade with vedolizumab. It remains to be elucidated which effects ontamalimab might have on innate immune cells, which is particularly important for understanding the results of the clinical trials. This study substantially increases our understanding of the specific mode of action of anti-trafficking therapies in IBD and might contribute to optimized future treatment strategies.
Summary
A fully functional neuritic network within the brain is essential for its integrity and a healthy life. Neurodegenerative diseases such as Parkinson’s disease (PD) and Huntington’s disease (HD) alter neuritic processes leading to psychiatric, motor, and cognitive impairments. Currently, no cure is available for either of these diseases. A better understanding of the underlying pathogenesis is there-fore crucial. Evaluating different disease models in vivo and in vitro mimicking important features of neurodegenerative diseases is a prerequisite to unravel processes interfering with the integrity of neuritic networks. In this thesis, the compensatory neuritogenesis within the striatum of a rat PD model and dysfunc-tional neuronal processes related to mutant huntingtin as well as a possible hun-tingtin lowering strategy were examined within primary HD-patient fibroblasts and patient-derived induced pluripotent stem cells (hiPSCs), neural progenitor cells (NPCs), and cortical neurons.
As main hypothesis, we investigated the following: In vivo and in vitro models of neuronal protein aggregation disorders present a powerful platform to reveal dis-ease specific compensatory mechanisms and to assess therapeutic targets. The aims were divided investigating PD and HD in an in vivo and in vitro model, re-spectively. Thus, the first aim of the present thesis is to examine whether the serotonergic input towards the dorsal striatum is altered due to the reduced do-paminergic input in a transgenic rat PD model. The second aim investigates whether mutant huntingtin causes dysfunctional neuronal processes and investi-gates a possible pharmacological intervention in primary HD-patient fibroblasts and patient-derived hiPSCs, NPCs, and cortical neurons.
Addressing the first aim, a transgenic rat PD model ubiquitously expressing hu-man α-synuclein using a bacterial artificial chromosome (BAC hα-syn tg rat) was used. The dopaminergic and serotonergic neuritic network was analyzed within the dorsal striatum, motor cortex M1/M2, raphe nuclei, and substantia nigra pars compacta of 12-month-old BAC hα-syn tg and non-tg rats. Structural analysis of the dopaminergic innervation showed a significant decrease in dopaminergic fi-ber density within the dorsal striatum of BAC hα-syn tg rats. Furthermore, the dopaminergic cell count within the substantia nigra was significantly reduced. In addition, a significant increase in serotonergic fiber density was detected by 100% within the dorsal striatum. In contrast, no structural changes of the dopaminergic and serotonergic innervation within the motor cortex M1/M2 were present. No changes in the number of serotonergic neurons and proximal fiber network within the dorsal and median raphe were observed. Immunofluorescence staining and imaging of transporter and enzymes important for L-DOPA uptake, dopamine synthesis, and dopamine release revealed the potential capacity of serotonergic neurons to synthesize dopamine from L-DOPA and its release. These findings indicate a site-specific compensatory neuritogenesis of serotonergic afferents within the dorsal striatum of BAC hα-syn tg rats. Together with the capacity of serotonergic neurons to synthesize and release dopamine, this might play a role in L-DOPA induced dyskinesia.
Addressing the second aim, an in vitro HD model, including HD-patient-derived fibroblasts, hiPSCs, NPCs, and cortical neurons, was established. HiPSCs showed a significant increase in NANOG expression while other pluripotency markers displayed no differences between HD and control indicating pathophysi-ological changes already at a stem cell level in cells derived from HD-patients. No differences in proliferation marker Ki67 and neural marker βIII-tubulin and CTIP2 expression were observed upon cortical neuron differentiation between HD-patients and controls. Furthermore, the expression of huntingtin was ana-lyzed in the in vitro HD model. No differences in total huntingtin levels were ob-served between HD-patient and control-derived fibroblasts, hiPSCs, NPCs, and cortical neurons. Mutant huntingtin was solely detected in HD-patient-derived cells. Treatment of HD-patient and control-derived fibroblasts and hiPSCs using Branaplam, a small molecule splice modulator, accomplished a profound reduc-tion in total and mutant huntingtin. This finding validates the in vitro HD model as a powerful screening platform for huntingtin lowering compounds and Branaplam as a potential compound to lower huntingtin levels in HD-patients.
These key findings demonstrate that in vivo and in vitro disease models are a powerful prerequisite for understanding underlying neurodegenerative processes in PD and HD. Thus, underlying pathogenic mechanisms are uncovered and new treatment strategies maybe tested in these models.
Tumor assoziierte Fibroblasten (engl.: cancer-associated fibroblasts; CAFs) sind ein wesentlicher Bestandteil des Tumormikromilieus und spielen eine bedeutende Rolle bei der Entstehung und Progression von Tumoren. Die Zytokine IL-6 und IL-11 sind Aktivatoren des Transkriptionsfaktors STAT3 (engl.: signal transducer and activator of transcription 3) und häufig im kolorektalen Karzinom (KRK) hochreguliert. Die IL-6- und IL-11-induzierte STAT3-Aktivierung in Tumorepithelzellen ist an der Pathogenese des KRK beteiligt. Allerdings war die Rolle der STAT3-Aktivierung in CAFs während der kolorektalen Karzinogenese nicht verstanden und wurde deshalb im Rahmen dieser Arbeit untersucht. Dazu wurde die pSTAT3-Expression in CAFs im Tumorgewebe von 375 Patienten mit einem KRK, unter Verwendung eines tissue-microarray, Immunfluoreszenzfärbung und digitaler Pathologie untersucht. Um die funktionelle Rolle der STAT3-Aktivierung in CAFs während der kolorektalen Karzinogenese zu untersuchen, wurden verschiedene murine in vivo Tumormodelle verwendet und mit modernen ex vivo Imaging-Verfahren kombiniert. Zur Untersuchung des Mechanismus, durch den die STAT3-Aktivierung in Kolonfibroblasten die Tumorgenese beeinflusst, wurde eine Genexpressionsanalyse mittels RNA-Sequenzierung von verschiedenen Fibroblasten-Subpopulationen (COL6+ vs. COL6-) nach STAT3-Aktivierung (IL-6 vs. IL-11) durchgeführt.
Die Analyse der pSTAT3-Expression in CAFs des humanen tissue-microarray zeigte eine inverse Korrelation der pSTAT3-Expression in CAFs mit dem Überleben der KRK-Patienten. In loss-of-function- und gain-of-function-Experimenten mit transgenen Mäusen, die eine Kollagen-VI- oder Kollagen-I-spezifische Modifikation der STAT3-Funktion aufweisen, konnte eine kritische Rolle der STAT3-Aktivierung in CAFs für die kolorektale Tumorentstehung im AOM/DSS-Modell aufgezeigt und in zwei weiteren Tumormodellen bestätigt werden. Durch den Vergleich der verschiedenen Genexpressionsprofile der Fibroblasten-Subpopulationen nach STAT3-Aktivierung konnte eine Überlappung der Profile, sowie die Regulation von Angiogenese-assoziierter Transkriptionsmuster aufgedeckt werden. Zudem reduzierte die Blockade pro-angiogener Signalwege das Wachstum kolorektaler Tumore bei Mäusen mit konstitutiver STAT3-Aktivierung in COL6+ Fibroblasten. Insgesamt konnte in dieser Arbeit eine kritische Rolle der STAT3-Aktivierung in CAFs für die kolorektale Karzinogenese gezeigt werden. Somit könnten Strategien, die die Aktivierung von STAT3 in CAFs hemmen, zukünftig zur Behandlung des KRK verwendet werden. Zudem könnte die Aktivierung von STAT3 in CAFs als prognostischer Marker bei KRK-Patienten dienen.
Professor Jens Kossmann was the Specialty Chief Editor of the Plant Biotechnology section at Frontiers in Plant Science from its inception until his death in March 2023 at the age of 59. He ran the section with great enthusiasm and was an important driver of its success. This obituary is designed to celebrate his contribution to plant science as well as his role as a mentor and friend.
The CD83 molecule is a powerful immunomodulatory protein which contributes to the resolution of inflammation. Expression of CD83 is a hallmark of mature dendritic cells (DCs), but this molecule is also expressed on other activated immune cells including B cells, T cells, regulatory T cells (Tregs), macrophages (Mφ) and on thymic epithelial cells (TECs). Two naturally occurring isoforms of this protein exist: a membrane-bound form (mCD83) and a soluble form (sCD83), which is the extracellular domain of the mCD83 protein. The soluble CD83 protein has profound pro-resolving functions, since administration of sCD83 in specific autoimmune disease and transplantation models, leads to reduction of disease severity on the one hand and on the other hand sCD83 application in transplantation significantly prolonged allograft survival by Treg induction. Conclusively, sCD83 is a potential candidate for new therapeutical treatment options for future clinical trials in the field of autoimmunity and transplantation.
Recent studies elucidated the biological role of the mCD83 protein on specific cell types, including DCs and Tregs, using conditional KO mice (cKO). Mice with CD83-deficiency on Tregs as well as DCs developed severe autoimmune reactions in the animal model for multiple sclerosis – i.e. the experimental autoimmune encephalomyelitis (EAE) model - compared to control animals. Therefore, the function of CD83 on Tregs as well as DCs indicates a similar pro-resolving function, as reported for the soluble CD83 protein (sCD83).
The first aim within the present study was to establish a new therapeutic concept of transplant-mediated tolerance induction by the sCD83 protein, within the high-risk cornea transplantation model, in cooperation with the Cornea Lab, Department of Ophthalmology, University Hospital of Cologne (PD Dr. rer. nat. Felix Bock and M.Sc. Alfrun Schönberg). Importantly, we show that sCD83 preincubation of donor-corneal allografts induces tolerogenic mediators within the graft recipients and prolongs graft survival. Preincubation of donor corneal tissue with sCD83 led to induction of Tregs, regulatory DCs as well as a shift from pro-inflammatory, classically activated Mφ (CAM) towards pro-resolving, alternatively-activated Mφ (AAM), in eye draining lymph nodes of graft recipients. Therefore, we further analyzed the impact of the sCD83 protein on Mφ and DC differentiation. Although previous studies reported an immunoregulatory function of sCD83 on the transition from iDCs to mDCs, there are no studies investigating whether sCD83 affects the monocyte to DC differentiation processes. Intriguingly, sCD83 induced a tolerogenic DC phenotype, which was characterized by low expression of costimulatory molecules, e.g. CD80 and CD86, whilst pro-resolving molecules including CD200R and Msr-1 were upregulated. MLR assays revealed a less stimulatory capacity of allogeneic T cells in the presence of sCD83-differentiated DCs. The phenotype of regulatory DCs was also confirmed by our collaborators, using the corneal transplantation model in vivo. Importantly, next to DCs, also Mφ play an important role in the modulation of the alloimmune response and there are no studies regarding the effect of sCD83 on Mφ. Therefore, we assessed the effect of sCD83 on Mφ and revealed that sCD83 induces alternatively activated Mφ (AAM)-like phenotypic and functional changes, which are able to induce Tregs.
Within the second aim of this study, the biological role of the mCD83 protein on Mφ was analyzed using conditional KO mice. First we assessed the expression of CD83 on these cells, using different pro- and anti-inflammatory stimuli, to generate two distinct Mφ populations: the pro-inflammatory, classically-activated Mφ (CAM) and pro-resolving, alternatively-activated Mφ (AAM), which have different phenotypes and functions. CD83 was stably and significantly upregulated on IL-4-stimulated pro-revolving AAM, while upon stimulation with pro-inflammatory stimuli such as LPS, Zymosan, TNF-α and IFN-ɣ, CD83 was only transiently upregulated. Furthermore, we found out, that CD83 is upregulated during the resolution of inflammation, indicating a pro-resolving function of CD83 expressed by Mφ.
To further characterize the functional relevance of CD83 on Mφ, we crossed CD83-floxed mice with CX3CR1-cre mice, to generate the conditional KO of CD83 on CX3CR1+ myeloid cells. Interestingly, deletion of CD83 results in a downregulation of MHCII and CD86 on Mφ, which is in line with previous studies investigating B cells as well as DCs. Moreover, we show that CD83 deletion results in a defect in the AAM phenotype and function. IL-4 stimulated CD83-deficient Mφ show an overactivated phenotype, which is characterized by reduced CD200R as well as Msr-1 expression levels and in line with that, we observed increased proinflammatory cytokine secretion in CD83-deficient AAM, including TNF-α, IL-6, CXCL1 and G-CSF. When we analyzed the phagocytic activity – a hallmark of AAM - CD83-deficient AAM showed a striking decrease in the engulfment of E.coli bacteria, which correlates with the observed reduced Msr-1 expression. When we analyzed the ability to kill E.coli, by unstimulated CD83-deficient Mφ, we observed an increase in their killing capacity, which was attributed to an increased ROS production. Moreover, when we cocultured CD83-deficient Mφ with allogeneic splenocytes in mixed lymphocyte reaction (MLR) assays, T cells proliferated stronger and proinflammatory cytokine expression levels were increased, whilst frequencies of Tregs were significantly reduced.
Taken together, within the present study, we established a new therapeutic approach in transplant mediated tolerance induction, using the high-risk cornea transplantation model, by preincubation of the donor tissue with sCD83. In addition, sCD83 modulates Mφ towards a pro-resolving AAM-like phenotype, whilst mCD83 deletion modulates Mφ towards a proinflammatory phenotype.
Background:
Tinnitus is an auditory phantom percept in the absence of external sound sources. Despite the high prevalence and tinnitus-associated distress of affected patients, the pathophysiology of tinnitus remains largely unknown, making prevention and treatments difficult to develop. In order to elucidate the pathophysiology of tinnitus, animal models are used where tinnitus is induced either permanently by noise trauma or transiently by application of salicylate. In a model of trauma-induced tinnitus, we have suggested a central origin of tinnitus-related development of neuronal hyperactivity based on stochastic resonance (SR). SR refers to the physiological phenomenon that weak sub-threshold signals for given sensors (or synapses) can still be detected and transmitted if appropriate noise is added to the sensor’s input. It is not clear if different tinnitus inducers lead to the same neurophysiological mechanisms of development, representation and perception of tinnitus. Even though the noise trauma model is the most common animal model of tinnitus induction, the salicylate model has several advantages, for example fast induction and a transient percept. If noise trauma and salicylate models would share the same mechanism of development and perception of tinnitus, tinnitus research could focus more on the salicylate model than the noise trauma model. The main objective of this study was to characterize the behavioral and neurophysiological effects like spatio-temporal cortical activity during salicylate-induced tinnitus and compare them with data from trauma induced tinnitus.
Material and methods:
Two different doses of salicylate were used for tinnitus induction in this dissertation (150 mg/kg and 300 mg/kg). The gap prepulse inhibition of the acoustic startle reflex (GPIAS) and auditory brainstem responses (ABR) were used to measure the presence of behavioral tinnitus signs and hearing loss (HL) after salicylate injection, respectively. Acute recordings of the local field potentials (LFP) in the auditory cortex (AC) were used to compute the spatio-temporal cortical activity before and after salicylate injection.
Results:
In line with the pharmacokinetics, hearing thresholds (HT) generally increased 2 h after salicylate injections. This increase was significantly stronger within the region of best hearing compared to other frequencies. Furthermore, animals showed behavioral signs of tinnitus during that time window and frequency range as assessed by GPIAS.
In contrast to animals with noise trauma induced tinnitus, salicylate-induced tinnitus animals showed no correlation between hearing threshold changes and behavioral signs of tinnitus, indicating that the development of tinnitus after salicylate injection is not based on SR, the mechanism proposed for the trauma model.
The cortical recordings on the other hand show some similarities with the noise trauma-induced tinnitus. The difference between the different frequency-specific and tinnitus-associated spatio-temporal activity patterns was reduced after both types of tinnitus induction. However, the activity pattern recorded during the“silent” condition, in which the tinnitus percept should be most prominent, becomes more similar to one specific frequency representation (specifically 4 kHz) in noise trauma animals, while in the salicylate model all representations collapse to one common range of patterns.
Conclusion:
In conclusion, I found that salicylate can induce tinnitus in our animal model but is not based on the same neurophysiological mechanism (SR) as noise trauma-induced tinnitus. Furthermore, the quality of the tinnitus percept as assessed by spatio-temporal cortical activity patterns is likely to be a pure tone in case of noise trauma animals and a rather broadband percept in salicylate animals. Therefore, it is not reasonable to replace noise trauma model with salicylate model, despite the technical advantages.
<p xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H<sup>+</sup> symporters. Apart from <italic>AtSUC2</italic>, which is companion cell specific, all other <italic>AtSUC</italic> genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an <italic>Atsuc1 knockout</italic> plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line <italic>Atsuc1Atsuc3Atsuc8Atsuc9</italic> shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that <italic>AtcwINV2</italic> and <italic>AtcwINV4</italic> are expressed in pollen, <italic>AtcwINV1</italic> in the transmitting tissue and <italic>AtcwINV5</italic> in the funiculi of the ovary. The vacuolar invertase <italic>AtVI2</italic> is also expressed in pollen, and a knockout of <italic>AtVI2</italic> leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.</p>
The lung disease tuberculosis (TB) caused by the intracellular bacterium Mycobacterium tuberculosis (MTB) remains the deadliest infectious disease worldwide. Macrophages are the host cells of mycobacteria and are the first to come into contact with the pathogen after inhalation. They express the DECTIN-2 family C-type lectin receptors (CLRs) MINCLE, MCL and DECTIN-2 which recognize several mycobacterial ligands including Trehalose-6,6-dimycolate (TDM) and its synthetic analog Trehalose-6,6-dibehenate (TDB). Activated CLRs trigger a signal cascade that ultimately induces inflammatory gene expression in innate immune cells and contributes to anti-mycobacterial immunity. Additionally, MINCLE is crucial for effective adjuvant responses through TDB and thus contributes to vaccine responses. Expression and function of CLRs are thereby regulated by different key cytokines including Interleukin-4 (IL-4) or Tumor necrosis factor (TNF) that can either dampen or promote CLR signaling and thus influence mycobacterial infections. This study aims to address the impact of TNF and IL-4 on CLR expression and function regarding myeloid cell activation and vaccination.
In the first part, we show that TNF was sufficient to induce Mincle, Mcl, and Dectin-2 expression in bone marrow-derived macrophages (BMDMs). TNF-TNFR1-p55 signaling was required for upregulation of these CLRs and for cytokine secretion upon stimulation with TDB or Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Importantly, in vivo immunization experiments using the MINCLE-dependent TDB-containing liposomal adjuvant CAF01 identified TNF to be essential for TH17 responses since TNF-deficiency or blockade of TNF with etanercept abrogated these responses. Thus, blocking of TNF or interference with TNF signaling pathways counteract DECTIN-2 family CLR expression which may inhibit vaccine responses and potentially enhance the risk of mycobacterial infection.
In the second part, we show that IL-4 downregulated Mincle, Mcl, and Dectin-2 gene expression as well as pro-inflammatory cytokine production in BMDMs in response to BCG in vitro. To investigate the effect of high levels of IL-4 on MINCLE expression and function in vivo, we made use of a plasmid-driven overexpression system and TH2-biased helminth co-infection models. BCG infection in mice induced MINCLE surface expression on peritoneal monocytes but not neutrophils, which was inhibited by systemic overexpression of IL-4 or by co-infection with Nippostrongylus brasiliensis (N.b.). Functionally, phagocytic uptake of BCG by BMDMs was neither affected by IL-4 in vitro, nor by N.b. co-infection in vivo. Importantly, TH1/TH17 differentiation induced by CAF01 was inhibited in the spleen, but not in the draining lymph nodes of mice infected with N.b. or Schistosoma mansoni (S.m.). These results provide new insights into how helminth co-infections can interfere with CLR-dependent signaling thereby thwarting anti-mycobacterial immunity and MINCLE-dependent vaccination responses.
Despite improvements in prevention and treatment approaches, colorectal cancer (CRC) still remains a highly life-threatening disease worldwide. The formation of metastasis, in particular in the peritoneal cavity, presents one of the most fearsome aspects due to poor prognosis and limited therapeutic strategies. Deciphering the molecular mechanisms of transcoelomic dissemination and identification of novel biomarkers for metastatic spread towards the peritoneum are highly anticipated to improve screening approaches and overall survival.
The activating transcription factor 2 (ATF2) exhibits a highly antagonistic character since it can either function as tumor suppressor or oncogene in a context- and stimulus-dependent manner. As the role of ATF2 in CRC has been controversially discussed, this PhD thesis aimed to elucidate the function of ATF2 in CRC using in vitro, in vivo and translational approaches to unravel novel putative biomarkers for CRC treatment.
Immunohistochemical evaluation of ATF2 expression in human colon cancer tissues using a tissue microarray demonstrated that reduced ATF2 levels were associated with poor overall survival, suggesting a tumor suppressive function for ATF2 in CRC. Intriguingly, enormous intratumoral heterogeneity (ITH) for ATF2 was detected at tumor invasion fronts. Monoclonal ATF2 knockout (KO) clones were generated in HCT116 and HT29 cells using CRISPR/Cas9 gene editing to abrogate ITH. NanoString gene expression analysis identified a remarkable upregulation of tumor-associated calcium signal transducer 2 (TACSTD2) within a unique ATF2 loss gene signature. Modulation of mitogen-activated protein kinases provided further evidence for a negative correlation between ATF2 and TACSTD2. Given the presence of AP-1 binding sites in the TACSTD2 promoter, chromatin immunoprecipitation was performed, suggesting TACSTD2 as a potential novel ATF2 target gene. Functional characterization of ATF2 KO cells revealed a TACSTD2-mediated de-adhesive phenotype as suggested by filopodia formation in confocal microscopy and reduced aggregate forming capacity in detachment assays which could be both reverted upon transient TACSTD2 silencing. Consistently, the de-adhesive phenotype was reflected in vivo in a chorioallantoic membrane (CAM) model in which ATF2-deficient cells formed loosely packed tumor masses. Loss of ATF2 appeared to alter the invasion pattern of tumor cells as shown in vitro and in vivo, resulting in a deeper muscle invasion in a subcutaneous mouse xenograft model. Analysis of CAM invasion fronts revealed low-proliferative ATF2-negative cell clusters with enhanced membranous E-Cadherin/β-Catenin expression, a phenotype reminiscent of leader cells.
This PhD thesis revealed distinct ATF2 expression patterns dependent on the tumor topology, suggesting TACSTD2-mediated de-adhesion in the tumor center as the first step of the metastatic cascade, and the formation of aggressive leader cells at the invasion front. This minor ATF2-negative subpopulation might act as a potential driver for collective invasion and high tumor aggressiveness. In conclusion, ATF2 could serve as a putative biomarker for aggressive leader cells and peritoneal metastasis, which could be further investigated using the newly established conditional Atf2 knockout mouse strain in appropriate CRC in vivo models.
Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 µm wide microfluidic channel. The fluid shear stress induces large, ear ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe [1] that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell-cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.
Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein–Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.
Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.
Functionality of the native tissue is a result of complex interaction between cells and extracellular
matrix (ECM) and depends on the composition, architecture and mechanical properties of the
ECM, as well as the adequate supply of oxygen and nutrients. Advanced biofabrication techniques
offer a great possibility to recapitulate these functional features in the laboratory. However,
creating functional tissue models with appropriate ECM characteristics and adequate support of
tissue specific cell types remains challenging. The presented work aimed at developing
physiologically-relevant, endothelialized microvacular networks embedded within an engineered
matrix. To achieve this, analyses related to microchannel endothelialization and cell-matrix
interactions were conducted.
Completing the thesis within the SFB-TRR225 consortium enabled me to perform collaborative
studies at the interface of biology, chemistry and engineering. In the first part of my thesis,
sacrificial scaffolds produced from thermoresponsive polymer poly(2-oxazoline) (POx) using melt
electro writing (MEW) technique were used. Bifurcated sacrificial scaffolds were either directly
seeded with primary human endothelial cells (ECs) for endothelial layer formation on their surface,
or were embedded in bulk hydrogels placed in a customized bioreactor. In the latter approach, POx
was removed by temperature lowering to create bifurcating microchannels, which were
subsequently endothelialized and exposed to bidirectional perfusion. The second approach enabled
a rapid formation of a tight and biologically functional endothelial layer.
In the second part of my thesis, different hydrogels were evaluated with regard to their suitability
as matrices for microvascularized models and tissue engineering. Extensive analyses were done to
determine their biocompatibility with ECs and primary fibroblasts. Within those, oxidized
alginate-based hydrogels (ADA and ADA-GEL), as well as allyl-modified gelatin (gelAGE), were
casted with primary cells and investigated using flow cytometric, spectrometric, as well as imaging
techniques. Molecular studies with ADA and ADA-GEL demonstrated the mechanisms of their
toxicity towards ECs and of fibroblast survival. GelAGE hydrogels were tailored to improve the
response of ECs and fibroblasts and were shown to support cell viability, motility, and biological
functions.
The results obtained within this thesis are of high importance for the progress of the SFB-TRR225
within the cross-cutting topics of vascularization and tumor models, as well as for the field of
biofabrication. The developed endothelialized microvascular networks, together with the suitable
7
ECM candidates will be in the future tailored to the needs of specific tissue models for the purpose
of disease modelling and drug screening studies.
Abstract
Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants.
Abstract
Erysimum crepidifolium Rchb. is one of the few Brassicaceae species accumulating glucosinolates as well as cardenolides. This is possibly providing a selective advantage in evolution as both compounds are part of a chemical defense system. In order to study the biosynthesis of these compounds, a regeneration protocol for E. crepidifolium using in vitro shoot cultures derived from seeds has been developed. Murashige and Skoog (MS) culture medium supplemented with various combinations of cytokinins and auxins was used. MS medium containing NAA (naphthaleneacetic acid, 0.04 mg mL−1) and BAP (6-benzylaminopurine, 0.2·10−2 mg mL−1) proved to be optimal for root formation. Plantlets developed well on modified MS medium without the use of phytohormones. About 80% of the plantlets rooted in vitro developed into intact plants after transfer to the greenhouse. Cardenolides (1.75 mg g−1 dry weight (DW)) were detected in cultured shoots on solid DDV media while glucosinolates mainly accumulated in roots where 0.025 mg g−1 FW were detected in shoots cultured on the same medium (DDV). The expression of two progesterone 5β-reductase and three Δ5-3β-hydroxysteroid dehydrogenase genes were measured in shoot cultures since the encoded enzymes are supposed to be involved in cardenolide biosynthesis. E. crepidifolium shoot cultures propagated on solid media meet the necessary requirements, i.e., clonal homogeneity, product accumulation, and gene expression, for a suitable model to study cardenolide but not glucosinolate biosynthesis.
Synthetic nanoparticles functionalized with stabilized trimers of the HIV-1 envelope glycoprotein (Env) have become a major focus in AIDS vaccine research. The additional incorporation of T helper cell epitopes into such nanoparticles might recruit pre-existing CD4+ T cell responses induced by childhood vaccinations to provide intrastructural help (ISH) for Env-specific B cells upon immunization. In this study, calcium phosphate nanoparticles (CaPs), that encapsulate an immunodominant Tetanus Toxoid epitope (p30) and display Env trimers on the surface (T helper CaPs), were evaluated in preclinical vaccine trials. T helper CaPs induced the activation of Env-specific naïve B cells in vitro, which was not observed with soluble Env trimers. Immunization with T helper CaPs resulted in significantly stronger Env-specific humoral immune responses via ISH in mice that were immunized in advance with a licensed Tetanus vaccine. The magnitude of anti-Env antibody levels in ISH mice was comparable to a control group immunized with CpG-adjuvanted CaPs. In contrast to CpG, the induction of immune mechanisms suspected to increase the susceptibility for HIV infection was bypassed by harnessing ISH. Having provided evidence that the utilization of T helper CaPs resulted in versatile, immunomodulatory features, the nanoparticle design was improved by an orthogonal Env coupling mechanism (oCaPs). To this end, a genetically encoded aldehyde-tag (LCTPSR) was introduced at the C-terminus of native-like, soluble Env trimers. The tag-associated cysteine is post-translationally converted into a formylglycine harboring an aldehyde group, which was confirmed by mass spectrometry. This aldehyde was used for covalent bioconjugation with an aminooxy/alkyne-crosslinker. Linker-bound Env trimers (lnkr-Env) were then immobilized on the CaP surface via a Click reaction. Reporter assays based on fluorescent gel analysis and CLSM proved that the Env conjugation was highly aldehyde-specific and efficient. Most importantly, conformational ELISA and surface FACS analyses indicated that the pre-fusion conformation is preserved. oCaPs induced stronger B cell activation in vitro and higher Env-specific antibody levels in vivo than randomly coupled CaPs. Taken together, this study established a nanoparticle delivery platform for future immunomodulatory vaccine approaches and diagnostic applications in the context of various pathogens.
Plectin, a highly versatile cytolinker protein, is crucial for myofiber integrity and function. Accordingly, mutations in the human gene (PLEC) cause several rare diseases, denoted as plectinopathies, with most of them associated with progressive muscle weakness. Of several plectin isoforms expressed in skeletal muscle and the heart, P1d is the only isoform expressed exclusively in these tissues. Using high-resolution stimulated emission depletion (STED) microscopy, here we show that plectin is located within the gaps between individual α-actinin-positive Z-disks, recruiting and bridging them to desmin intermediate filaments (Ifs). Loss of plectin in myofibril bundles led to a complete loss of desmin Ifs. Loss of Z-disk-associated plectin isoform P1d led to disorganization of muscle fibers and slower relaxation of myofibrils upon mechanical strain, in line with an observed inhomogeneity of muscle ultrastructure. In addition to binding to α-actinin and thereby providing structural support, P1d forms a scaffolding platform for the chaperone-assisted selective autophagy machinery (CASA) by directly interacting with HSC70 and synpo2. In isoform-specific knockout (P1d-KO) mouse muscle and mechanically stretched plectin-deficient myoblasts, we found high levels of undigested filamin C, a bona fide substrate of CASA. Similarly, subjecting P1d-KO mice to forced swim tests led to accumulation of filamin C aggregates in myofibers, highlighting a specific role of P1d in tension-induced proteolysis activated upon high loads of physical exercise and muscle contraction.
Abstract
Pathogenic bacteria possess a great potential of causing infectious diseases and represent a serious threat to human and animal health. Understanding the molecular basis of infection development can provide new valuable strategies for disease prevention and better control. In host‐pathogen interactions, actin‐cytoskeletal dynamics play a crucial role in the successful adherence, invasion, and intracellular motility of many intruding microbial pathogens. Cortactin, a major cellular factor that promotes actin polymerization and other functions, appears as a central regulator of host‐pathogen interactions and different human diseases including cancer development. Various important microbes have been reported to hijack cortactin signaling during infection. The primary regulation of cortactin appears to proceed via serine and/or tyrosine phosphorylation events by upstream kinases, acetylation, and interaction with various other host proteins, including the Arp2/3 complex, filamentous actin, the actin nucleation promoting factor N‐WASP, focal adhesion kinase FAK, the large GTPase dynamin‐2, the guanine nucleotide exchange factor Vav2, and the actin‐stabilizing protein CD2AP. Given that many signaling factors can affect cortactin activities, several microbes target certain unique pathways, while also sharing some common features. Here we review our current knowledge of the hallmarks of cortactin as a major target for eminent Gram‐negative and Gram‐positive bacterial pathogens in humans.
Abstract
The bottom‐up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA‐encoded informational content are still in their infancy. Herein, droplets of DNA Y‐motifs are formed by liquid–liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y‐motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio‐temporally controlled DNA segregation in bulk as well as in cell‐sized water‐in‐oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice‐based theoretical model which mimics the interactions between the DNA Y‐motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom‐up assembled synthetic cells.
Microgravity (µg) has a massive impact on the health of space explorers. Microgravity changes the proliferation, differentiation, and growth of cells. As crewed spaceflights into deep space are being planned along with the commercialization of space travelling, researchers have focused on gene regulation in cells and organisms exposed to real (r-) and simulated (s-) µg. In particular, cancer and metastasis research benefits from the findings obtained under µg conditions. Gene regulation is a key factor in a cell or an organism’s ability to sustain life and respond to environmental changes. It is a universal process to control the amount, location, and timing in which genes are expressed. In this review, we provide an overview of µg-induced changes in the numerous mechanisms involved in gene regulation, including regulatory proteins, microRNAs, and the chemical modification of DNA. In particular, we discuss the current knowledge about the impact of microgravity on gene regulation in different types of bacteria, protists, fungi, animals, humans, and cells with a focus on the brain, eye, endothelium, immune system, cartilage, muscle, bone, and various cancers as well as recent findings in plants. Importantly, the obtained data clearly imply that µg experiments can support translational medicine on Earth.