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Aortic valve (AV) and root replacement with composite graft and re-implantation of coronary arteries described first by Bentall and de Bono in 1968, is considered as a standard operation for treatment of different pathologies of the AV and aortic root. In centres where aortic valve and root repair techniques and Ross operation are well established, generally severely diseased patients remain indicated for this procedure. The aim of this study was to evaluate the early and long-term outcomes after Bentall-De Bono (BD) procedures in high-risk population with complex pathologies and multiple comorbidities.
Between 2005 and 2018, a total of 273 consecutive patients (median age 66 years; 23 % female) underwent AV and root replacement with composite-graft in so called button technique. We divided our population in the following groups: 1. acute type A aortic dissection group (ATAAD) (n = 48), 2. endocarditis group (n = 99) and 3. all other pathologies group (n = 126). The surgery has been per- formed emergent/urgent in 131 patients (49 %) and in 109 cases (40%) as a reoperation. Concomitant surgery was required in 97 patients (58%) and 167 pa- tients (61%) received a biological composite-graft.
Follow-up was completed in 96% (10 patients lost to follow-up) with a mean of 8.6 years (range 0.1-15.7 years), counting a total of 1450 patient-years. Thirty- day mortality was 17% (46 patients). The overall estimated survival in 5 and 10 years was 64% ± 3%) and 46% ±4 %). Group comparison showed a significant difference in favour of patient from the dissection group (p = 0.008). Implantation of a biological valve graft was associated with lower survival probability (p < 0.001). There was no significant difference in the freedom of reoperation rate between the groups. The same applies for freedom of postoperative endocarditis, thromboembolic events, and aortic prosthesis dysfunction. According to the uni- variate and multivariate logistic regression analysis primarily postoperative neu- rological dysfunction (OR 5.45), hypertension (OR 4.8) peripheral artery disease (OR 4.4), re-exploration for bleeding (OR 3.37) and postoperative renal replace- ment therapy (OR 3.09) were identified as leading predictors of mortality.
In conclusion, the BD operation can be performed with acceptable short- and long-term results in high-risk patients with complex aortic pathologies in a centre with well-established AV repair and Ross operation program.
Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that typically begins in childhood and is associated with the cardinal symptoms of inattentiveness, hyperactivity, and impulsiveness. In a significant number of cases, ADHD persists into adulthood and leads to profound psychosocial impairment and costs to the population. The course of the disorder and the severity of psychosocial impairment are further influenced by the presence of comorbidities. The risk of developing psychiatric comorbidities such as affective disorders, personality disorders and substance use disorders is increased compared to the general population. Studies also indicate that ADHD is associated with a higher burden of somatic disorders such as obesity, diabetes mellitus, asthma and migraine. In the last decades, there has been a growing body of research that identified sex-related differences in ADHD, but there is still insufficient evidence on specific issues. In addition to the sex-ratio, which is more balanced in adulthood compared to childhood, there are also indications that differences exist at the symptom level and that the comorbid disorders that occur more frequently in ADHD also seem to differ in men and women, although the studies are not yet clear on this. Using resting-state analyses of functional magnetic resonance imaging (fMRI), we aimed to address the question of whether we can detect sex differences in ADHD and selected comorbidities (substance use disorder, depression, obesity) based on altered functional connectivity profiles. A central role for the pathogenesis of ADHD is the dysregulation of dopaminergic neurotransmission, specifically altered reward processing, as an expression of impaired impulse control. In the present study, we focused on a neuroanatomical hub, namely, the external part of the globus pallidus (GPe), which we defined as a "region of interest" for the analyses performed. There is growing evidence that the globus pallidus not only plays a role in the extrapyramidal motor system, but also integrates cognitive and reward-related information, functions that are impaired in ADHD. In a first step, we looked for sex differences in ADHD patients (n=137) and separately in healthy controls (HC) (n=45), then we compared a similar group of HC and ADHD patients to compare sex-differences in ADHD patients and HC. In a second step, we investigated whether the neural basis of comorbidity patterns differed between male and female patients. Analysis of the images of 182 participants was performed using the SPM-based CONN toolbox V 18.b. When comparing subjects with ADHD and HC, we observed an interaction between the GPe and the middle left temporal gyrus, with the effect being more pronounced in healthy subjects. When analyzing the large ADHD sample, an interaction between the GPe and the frontal pole/middle right frontal gyrus was observed. The connectivity between the GPe and the frontal and temporal brain areas appeared to be more pronounced in female ADHD patients than in males, with the sex-effect being reversed and more pronounced in healthy subjects. The results suggest that in patients with ADHD there is a loss of sex-specialization in GPe-connectivity. Males with ADHD and depression showed lower functional connectivity between the GPe and parts of the occipital cortex than females with ADHD and depression. To our knowledge, this is the first study to investigate sex-specific functional connectivity networks using a seed-based connectivity analysis of the external globus pallidus in adult ADHD patients with and without comorbidities. The study serves to improve our knowledge of GPe involvement in ADHD and sex-specific recruitment of this network. Taken as a whole, this study contributes to our understanding of the neurobiological correlates of ADHD and suggests possible differences between males and females with ADHD centered on altered connectivity with the GPe, helping to provide a different perspective on current research and new ideas for further studies.
Purpose: The aim of this work was to retrospectively identify prognostic factors for patients with neuroendocrine liver metastases (NELM) undergoing conventional transarterial chemoembolization (c-TACE), microwave ablation (MWA) or laser interstitial thermal therapy (LITT) and to determine the most effective therapy in terms of volume reduction and survival.
Method: Between 1996 and 2020, 130 patients (82 men, 48 women) were treated with c-TACE, 41 patients were additionally treated with thermoablative procedures.
Survival was retrospectively analyzed by using Kaplan-Meier-method. Prognostic factors were derived by using cox-regression. To find predictive factors for volume reduction due to c-TACE, a mixed-effects model was used.
Results: With c-TACE, an overall median volume reduction of 23.5 % was achieved. An average decrease of tumor volume was shown until the 6th c-TACE treatment, then the effect stopped. So, the median volume reduction off all lesions takes on a negative value from the 7th c-TACE intervention onwards. The mixed-effects model demonstrated that c-TACE interventions were most effective at the beginning of c-TACE therapy, and that treatment breaks longer than 90 days negatively influenced the outcome. For all patients evaluable for survival, Kaplan-Meier analysis showed a 1-year survival rate of 75 % and a 5-year survival rate of 36 %. Significant prognostic factors for survival were number of liver lesions (p = 0.0001) and therapeutical intention (p < 0.0001). Considering the clinical indication, 90.9 % of curative patients and 43.6 % of palliative patients responded to c-TACE therapy and thus could be submitted to a thermoablative procedure. Minor and one major complication occurred in 20.3 % of LITT and only in 8.6 % of MWA interventions. Complete ablation was observed in 95.7 % (LITT) and 93.1 % (MWA) of interventions
Conclusions: C-TACE is an effective treatment for volume reduction of NELM, however efficacy decreases after the 6th intervention and treatment breaks longer than 90 days should be avoided. With thermal ablation, a high rate of complete ablation was achieved and survival improved. Significant factors for survival were found and may be used as prognostic factors in the future.
The visual system encompasses about 20% of the cerebral cortex1 and plays a pivotal role in higher-order cognitive processes such as attention and working memory. Cognitive impairments constitute a central role in neuropsychiatric disorders such as schizophrenia (SZ). Impairments are described in visual perceptual processes including contrast, and emotion discrimination as well as in the ability to identify visual irregularities and in higher-order cognition like visual attention and working memory. Furthermore, perceptual and higher-order cognitive processes are part of the Research Domain Criteria (RDoC) project that aims to develop dimensional and transdiagnostic constructs with defined links to specific brain circuits.Therefore, the detailed study of the visual system using functional magnetic resonance imaging (fMRI) is essential to understand the processes in healthy individuals but also in populations with neuropsychiatric disorders. Visual mapping techniques include functional localizer tasks to map functionally defined regions like the fusiform face area (FFA), retinotopic mapping to map specific brain regions that are retinotopically organized in full, and visual-field localizer paradigms to define circumscribed areas within retinotopically organized areas.Thus, the latter allow studying local information processing in early visual areas. Despite advances in neuroimaging techniques, analyses of fMRI data at the group-level are impeded by interindividual macroanatomical variability. This reduces the reliability to accurately define visual areas particularly at the group-level and decreases statistical power. Single-subject based solutions for this problem are not appropriate. Analyses after volume-based alignment (VBA) and primary surface-based analyses without macroanatomical alignment do not increase macroanatomical correspondence sufficiently. Cortex-based alignment (CBA) approaches are recommended as an alternative technique to address this obstacle. However, CBA has not been evaluated for visual-field localizer paradigms. Therefore, we aimed to evaluate potential benefits of CBA for an attention-enhanced visual field localizer paradigm that maps circumscribed regions in retinotopically organized visual areas. Since previous studies solely compared surface-based data before and after CBA, we aimed to compare all three techniques: (1) a volume-based alignment (VBA), (2) a surface-based data set without (SBAV) and (3) a surface- based data set with macroanatomical alignment (CBA). Furthermore, we sought to define regions of interest (ROI) that subsequently can be used for the study of higher-order cognitive processes. Also, we aimed to investigate whether CBA facilitates the study of functional asymmetries in early visual areas as these were described in previous studies. Healthy volunteers (n=50) underwent fMRI in a 3- Tesla Siemens Trio scanner while performing an attention-enhanced visual field localizer paradigm. Our task consisted of a series of flickering, black-and white colored checkerboard stimuli that randomly appeared at one of four locations comprising the participants’ visual quadrants. In 25% of the trials the centrally located squares briefly changed their color to yellow (target trial). Participants had to indicate detection of a target by button press. Data analysis was conducted using Brain Voyager 20.6. Our approach for macroanatomical alignment included a high-resolution, multiscale curvature driven alignment procedure minimizing interindividual macroanatomical variability. Here, each folding pattern was aligned to a dynamically updated group average. Thus, we counteracted a possible confounding effect of a suboptimal selection of an individual target brain with a folding pattern deviating considerably from the cohort average. Group ROIs after CBA showed increased spatial consistency, vertical symmetry, and an increase of size. This was corroborated by an increase in the probability of activation overlap of up to 86%. CBA increased macroanatomical correspondence and thus ameliorated results of multi-subject ROI analyses. Functional differences in the form of a downward bias in visual hemifields were measured with increased reliability. In summary, our findings provide clear evidence for the superiority of CBA for the study of local information processing in early visual cortex at the group-level. This approach is of relevance for the study of visual dysfunction in neuropsychiatric disorders including schizophrenia as they show impaired visual processing that in turn impacts higher-order cognitive processes and in consequence functional outcome. In addition, our attention-enhanced visual field localizer paradigm will be useful for machine learning approaches such as multivariate pattern analysis decoding local information processes and connectivity patterns.
Cardiovascular disease (CVD) is the leading cause of death in the western world. Aging as the major risk factor for the development of CVD leads to structural changes in the heart and the vasculature. In addition to endothelial cells, mural cells, including smooth muscle cells and pericytes, form the vascular wall. Pericytes are defined as the perivascular cells located in the basement membrane of the capillaries, which are the smallest components of the vascular system and ensure the gas exchange in the tissue. In the different parts of the terminal vascular bed, pericytes receive different phenotypes and organ-specific functions. In addition to the stabilization of the vascular wall, pericytes are relevant for the formation of new vessels. Due to their potential of multipotent stem cells, pericytes can differentiate into different cell types and thus take a position in developmental processes. Pericytes play a crucial role in the development and diseases of the vascular system. Moreover, pericyte coverage is reduced in the aged heart. Nonetheless, the function of pericytes in the heart and their importance during cardiac aging is not completely understood.
To study the pericyte population in the aging heart, we have performed single-nucleus RNA-sequencing analysis comparing hearts from 12-weeks-old (young) and 18-month-old (old) mice. The detailed analysis of 336 differentially expressed genes (DEG) revealed that Rgs5 is downregulated in aged pericytes. Regulator of G-protein signaling 5 (RGS5), an established marker for pericytes, is involved the regulation of the blood pressure and in the formation of various cardiovascular diesases, including cardiac hypertrophy, myocardial infarction and atherosclerosis. We have furthermore confirmed this observation in vivo. Gene ontology (GO) analysis of DEG revealed that aged pericytes are characterized by the downregulation of genes involved in cell adhesion. Further, we have performed cell biology approaches using human brain vascular pericytes (hBVP) to investigate the role of Rgs5 in pericytes in vitro. Efficient knockdown of RGS5, although has no effect on cellular metabolism, viability and endothelial permeability, induces a reduction of pericyte adhesion to both a gelatine matrix and endothelial cells in a 3D matrigel culture. This was associated with the formation of filopodia. The altered phenotype suggested a changing identity of the pericytes. We could confirm that a loss of RGS5 causes a decreased expression of the pericyte markers PDGFRb and NOTCH3 and also leads to an overexpression of COL1A1, a fibroblast marker.
Together, our findings suggest that RGS5 is required for pericyte adhesion to endothelial cells and its downregulation in the aged mural cells could explain the reduction of pericyte coverage in the aged hearts. Further, RGS5 may be the key regulator for pericyte identity, as pericytes show an altered expression profile of cellular markers. The dedifferentiation of pericytes to a more fibroblast-like cell type could explain the increased fibrosis during age-related cardiac remodeling. We believe that RGS5 is a great candidate to explore and study the molecular mechanisms that regulate pericyte function in the heart, both in homeostasis and during aging.
G-protein-coupled receptors (GPCRs) comprise the largest transmembrane receptor family encoded in the human genome. GPCRs mediate the effect of a wide diversity of stimuli including light, odorants, ions, lipids, small peptides, and hormones. GPR182 is a GPCR for which no endogenous ligand has been identified yet. In the absence of an identified ligand, GPR182 remained poorly understood, and its biological functions had remained elusive. The presented work shows that GPR182 is highly and specifically expressed in microvascular endothelial cells. Phylogenetically, GPR182 is closely related to the atypical chemokine receptor 3 (ACKR3). Here, I show that GPR182 binds the chemokines CXCL10, -12 and -13. Similarly to other so-called atypical chemokine receptors, GPR182 is not coupled to G-proteins but is rather constitutively internalized following β-arrestin 2 recruitment. Consistent with potential scavenger functions, we detected increased concentration of the chemokines which bind the receptor in the plasma of Gpr182 deficient mice. Finally, we show that GPR182 plays an essential role in maintaining hematopoietic stem cells within the bone marrow niche. In summary, the data indicate that GPR182 is a novel member of the group of atypical chemokine receptors, which plays an important role in the chemokine/chemokine receptor network.
Slack (sequence like a Ca2+ -activated K + channel; also termed Slo2.2, Kcnt1, or KNa 1.1) is a Na+ -activated K + channel that is highly expressed in the peripheral and central nervous system. Previous studies have shown that Slack is enriched in the isolectin B4binding, non-peptidergic subpopulation of C-fiber sensory neurons and that Slack controls the sensory input in neuropathic pain. Recent single-cell RNA-sequencing studies suggested that Slack is highly co-expressed with transient receptor potential (TRP) ankyrin 1 (TRPA1) in sensory neurons. By using in situ hybridization and immunostaining we confirmed that Slack is highly co-localized with TRPA1 in sensory neurons, but only to a minor extent with TRP vanilloid 1. Mice lacking Slack globally or conditionally in sensory neurons (SNS-Slack─/─ ), but not mice lacking Slack conditionally in neurons of the spinal dorsal horn (Lbx1-Slack─/─ ), displayed increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. Patch-clamp recordings with cultured primary neurons and in a HEK-293 cell line transfected with TRPA1 and Slack revealed that Slack-dependent K + currents are modulated in a TRPA1-dependent manner. Taken together, these findings highlight Slack as a modulator of TRPA1-mediated activation of sensory neurons.
Furthermore, we investigated the contribution of Slack in the spinal dorsal horn to pain processing. Lbx1-Slack ─/─ mice demonstrated normal basal pain sensitivity and Complete Freund’s Adjuvant-induced inflammatory pain. Interestingly, we observed a significantly increased spared nerve injury (SNI)-induced neuropathic pain hypersensitivity in Lbx1-Slack ─/─ mutants compared to control littermates. Moreover, we tested the effects of pharmacological Slack activation in the SNI model. Systemic and intrathecal, but not intraplantar administration of the Slack opener loxapine significantly alleviated SNI-induced hypersensitivity in control mice, but only slightly in Lbx1Slack ─/─ mice, further supporting the inhibitory function of Slack in spinal dorsal horn neurons in neuropathic pain processing.
Altogether, our data suggest that Slack in sensory neurons controls TRPA1-induced pain, whereas Slack in spinal dorsal horn neurons inhibits peripheral nerve injury induced neuropathic pain. These data provide further insights into the molecular mechanisms of pain sensation.
Background and Aim: Genome-wide association studies revealed a strong association between cardiovascular diseases (CVD) and clonal hematopoiesis of indeterminate potential (CHIP), highlighting one of its most common CHIP-driving mutations-TET2 (ten-eleven translocation 2), as a target for CHIP related CVD research. Our lab has established the generation of self-organizing cardiac organoids (SCO), which demonstrate the cellular composition and organization of the native human heart, and mimics human myocardial responses to stress stimulation. This project aims to examine whether SCOs would be an appropriate CHIP model and decipher promising drugs for cardiovascular CHIP treatment.
Methods: To study TET2-mutant cardiovascular CHIP, we set up the TET2 cardiac-CHIP model through a knockdown (KD) of TET2 in myeloid cells that infiltrated our lab-made SCO. Immunofluorescence and qPCR were performed to ascertain TET2-KD myeloid cell infiltration, SCO fibrosis, and apoptosis assessments. SCO fibrosis was further analyzed by immunofluorescence staining, and cardiac contractile frequency and amplitude were determined by calcium flux analysis. Finally, RNAseq was performed to analyze transcriptomic changes in drug/vehicle-treated TET2-KD myeloid cells and the TET2 cardiac-CHIP model.
Results: The TET2 cardiac-CHIP model resulted in significantly increased inflammation in SCO, accompanied by fibrosis and more cleaved Caspase-3, causing cardiomyocytes apoptosis and promoting the release of cTNT. The shortlisted drugs revealed a reduction of proliferation in TET2-KD myeloid cells, decreased pro-inflammatory cytokines, and a higher apoptosis level. Furthermore, the TET2 cardiac-CHIP model treated with selected drugs showed a remarkable decline in TET2-KD myeloid cell infiltration and pro-inflammation cytokines, cardiomyocyte apoptosis, fibrosis, and lowered cTNT levels, while drug control groups were not affected. Moreover, the drug treatment groups improved the heartbeat frequency and amplitude accessed by the calcium transient assay. RNAseq data also validated the above findings.
Conclusions & Discussion: Our results indicate that SCOs are an efficient pre-clinical model for studying and validating CHIP genes and drug interactions. Our data revealed that TET2-KD myeloid cells invade SCO and secrete pro-inflammatory cytokines, which promote apoptosis of cardiomyocytes and the release of cTNT. In this regard, our TET2 cardiac-CHIP model matches the inflammatory phenotype previously characterized in CHIP patients. Nevertheless, this phenotype could be rescued using positive drug candidates (Clopidogrel, R406, and Lanatoside C) selected by this project, emphasizing the significant value of our TET2 cardiac-CHIP model for drug screens and pre-clinical validation studies. Furthermore, among these three drug candidates, we found Lancatoside C, as proved by FDA/EMA, showed an unmet possibility for clinical therapeutic demand, insinuating potential benefit in repurposing Lanatoside C for the treatment of TET2-mutant cardiovascular CHIP.
Background: In the Computer Tomography imaging, examinations for the diagnosis of lesions of the upper abdomen currently use water-soluble, iodinated, non-ionic contrast agents with low molecular weight. One possibility to reduce the time of the examination and X-ray exposure is to increase the injection rate. However, higher injections rates lead to increased hypersensitivity reactions and extravasation rates. Furthermore, cardiac pump function does not always allow for the transportation of such a large volume within one heartbeat. With a contrast agent of higher iodine concentration, the injection rate may be reduced without decreasing the iodine delivery rate while reducing the volume load of the heart.
Aim: to compare the performance and image quality of two injection protocols of contrast medium for multiphasic CT imaging of malignant hepatic lesions; one using Imeron 300 at an injection rate of 5 ml/ sec and the second using Imeron 400 at an injection rate of 3,7 ml/ sec, for multiphasic CT imaging of malignant hepatic lesions, in order to optimise the iodine concentration and injection rate of the contrast agent Imeron in the Multislice Spiral-CT of the upper abdomen.
Materials and methods: the current prospective, single centre, double-blinded, randomised and interindividual comparison study included 50 patients (29 males and 21 females) with a mean age of 63,3 years. Patients were randomised to one of the two injection protocols. Image evaluation included qualitative assessment (technical quality, presence of artefacts and overall contrast quality) and quantitative assessment (measuring the difference in HU between the lesion and the surrounding hepatic tissue). The difference between both protocols was tested for statistical significance using the Wilcoxon-Mann-Whitney test and the Two-Sample t-test.
Results: there was no statistically significant difference between both protocols regarding the technical quality of images, both in the AP (p = 0,46) and in the venous phase (p = 0,48). Additionally, no statistically significant difference was found regarding the presence of artefacts related to the contrast medium, both in the AP (p = 0,46) and in the venous phase (p = 0,46), as well as regarding the overall contrast quality of images both in the AP (p = 0,50) and in the venous phase (p = 0,48). Quantitative assessment showed no statistically significant difference regarding the difference in HU measurement between the hepatic lesion and the surrounding hepatic tissue, both in the AP (p = 0,36) and in the venous phase (p = 0,92).
Conclusion: in the multiphasic CT imaging of the liver, reducing the injection rate of the contrast medium Imeron from 5 ml/ sec to 3,7 ml/ sec while increasing the iodine strength of the agent from 300 to 400 mg/ml, respectively, and thus keeping the iodine injection flow rate constant, produces similar signal intensities and results in similar technical, image and overall contrast qualities..
Keywords: Contrast-medium, injection rate, iodine concentration, hepatic malignancy, multiphasic CT
The Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) as well as the T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) are rare types of malignant lymphomas. Both NLPHL and THRLBCL are frequently observed in middle-aged men with THRLBCL presenting frequently with an advanced Ann-Arbor stage with B-symptoms and associated with more aggressive courses.3 However, due to the limited number of tumor cells in the tissue of both NLPHL and THRLBCL, limited numbers of studies have been conducted on these lymphomas and current results are mainly based on general molecular genetic studies.
In order to obtain a better understanding for these disease forms as well as possible changes in their nuclear and cytoplasmatic sizes, the following study relied on the comparison of the different NLPHL forms and THRLBCL in terms of nuclear size and nuclear volume. This was carried out using both 2D and 3D analysis. During the 2D analysis of nuclear size and nuclear volume no significant differences could be presented between those groups. However, the 3D analysis of NLPHL and THRLBCL pointed out a slightly enlarged nuclear volume in THRLBCL. Furthermore, the analysis indicated a significantly increased cytoplasmatic size of THRLBCL compared to NLPHL forms. Nevertheless, differences occurred not only between the tumor cells of both disease forms, but also the T cells presented a larger nuclear volume in THRLBCL. B cells, which were considered as the control group, did not demonstrate any significant differences between the different groups. The presented results suggest an increased activity of T cells in THRLBCL, which is most likely to be interpreted as a response against the surrounding tumor cells and probably limits the proliferation of the tumor cells. Based on these results, the importance of 3D analysis is also evident due to the fact that it is clearly superior to 2D analysis. For a better understanding of both disease forms, it is therefore recommended to use the 3D technique in combination with molecular genetic analysis in future research.
Molecular oxygen (O2) is essential for numerous metabolic processes. Not surprisingly, hypoxia and the resulting adaptations play a pivotal role in pathophysiology, e.g., in cancer or in inflammatory diseases. Of note, myeloid cells are known to accumulate in hypoxic regions such as tumor cores or rheumatoid arthritis joints and may contribute to disease progression. While most studies so far concentrated on transcriptional adaptation by the hypoxia-inducible factors (HIF) 1 and 2 under short term hypoxia, prolonged oxygen deprivation and alternative post-transcriptional regulation are rather poorly investigated.
Consequently, the aim of the study was to generate a comprehensive overview of mRNA de novo synthesis and degradation and its contribution to total mRNA changes in monocytic cells in the course of hypoxia.
To this end, I used thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-Seq) to characterize RNA dynamics under hypoxia. Specifically, I labeled monocytic THP-1 cells under normoxia (N), acute hypoxia (AH; 8 h 1% O2), or chronic hypoxia (CH; 72 h 1% O2) with 4-thiouridine (4sU), which allows for transcriptome-wide identification of de novo synthesized mRNAs and estimation of their half-lives. Total mRNA expression analyses revealed that most changes occurred under CH. Considering that HIF accumulation and resulting transcriptional regulation was shown to decline again under CH, I further analyzed the impact of RNA stability on gene expression. I observed a global reduction in RNA half-lives under hypoxia, indicative for the attenuation of energy-consuming protein synthesis upon oxygen deprivation. Moreover, I observed a subgroup of hypoxic destabilized transcripts with resulting decreased mRNA expression under CH, which consisted of 59 nuclear-encoded mitochondrial mRNAs. This might prevent futile production of new mitochondria under conditions, where mitochondria are even actively degraded to prevent production of detrimental reactive oxygen species.
While stability-regulated transcripts were mainly destabilized under hypoxia, the vast majority of differentially de novo synthesized transcripts were upregulated.
Functional analyses revealed not only hypoxia, but also cholesterol homeostasis and inflammatory response as top enriched terms, corroborating findings on total mRNA level. Focusing on hypoxia-altered cholesterol metabolism, I observed an 9 accumulation of early and a decrease in late cholesterol precursors, which are separated by several oxygen-dependent enzymatic steps. Although total cholesterol levels were only slightly reduced, my data indicate locally lowered endoplasmic reticulum (ER) cholesterol levels under hypoxia, which cause feedback activation of the ER cholesterol-sensing transcription factor sterol regulatory element-binding protein 2 (SREBP2) and induction of cholesterol biosynthesis enzymes. Interestingly, a broad range of interferon-stimulated genes (ISGs), mainly known for their antiviral function, was also induced under hypoxia with similar kinetics as SREBP2 targets, suggesting an immunometabolic crosstalk. While the availability of certain cholesterol biosynthesis intermediates as well as a direct involvement of SREBP2 seemed rather unlikely to cause hypoxic ISG induction, changes in intracellular cholesterol distribution appeared crucial for the hypoxic induction of chemokine-ISGs. Mechanistically, I found that MyD88-dependent toll-like receptor 4 (TLR4) signaling contributes to enhanced hypoxic ISG induction, likely sensitized by changes in cholesterol dynamics. Importantly, hypoxia amplified induction of chemokine-ISGs in monocytes upon treatment with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein via TLR4 similarly as after addition of infectious virus, which might contribute to systemic inflammation in hypoxemic patients with severe coronavirus disease-2019 (COVID-19).
Taken together, I comprehensively analyzed RNA dynamics in hypoxic monocytes. Specifically, I identified RNA stability as a modulating mechanism to limit production of mitochondria under oxygen-restricted conditions. Moreover, I characterized the immunometabolic crosstalk between disturbed cholesterol homeostasis and spontaneous induction of interferon (IFN)-signaling in hypoxic monocytes, which might contribute to systemic inflammation in severe cases of COVID-19.
Aim: The cytochrome P450 reductase (POR) along with the cytochrome P450 enzymes (CYP) are responsible for the metabolism of a multitude of metabolites important for the maintenance of tissue function. Defects in this system have been associated with cardiovascular diseases. These enzymes are known to produce vasoactive lipids that modulate vascular tone. The aim of this study was to identify the consequence of a loss in endothelial POR for vascular function.
Methods and Results: To identify the endothelial contribution of the POR/CYP450 system to vascular function, we generated an endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-). Under basal condition ecPOR-/- already exhibited endothelial dysfunction in aorta and mesenteric vessels (acetylcholine-dependent relaxation, LogEC50 -7.6M for CTR vs. -7.2M for ecPOR-/- in aorta) and lower nitric oxide levels in the plasma (CTR: 236.8 ±77.4; ecPOR-/- 182.8 ±34.1 nmol/L). This dysfunction was coupled to attenuated eNOS function detected by the heavy arginine assay and decreased eNOS phosphorylation on S1177. Furthermore, insulin-induced phosphorylation of the eNOS activator, AKT, was also attenuated in the aorta from ecPOR-/- mice as compared to control mice. CYP450-dependent EET production was lower in plasma, lung and aorta of ecPOR-/- mice and this was accompanied with increased levels of vasoconstriction prostanoids (lipidomics of aorta, plasma and lung freshly isolated from CTR and ecPOR-/- mice). MACE-RNAseq from these aortas also showed a significant increase in genes annotated to eicosanoid production. In an in vivo angiotensin II model, acute deletion of POR increased the blood pressure as measured by telemetry and tail cuff (137.4 ± 15.9 mmHg in WT; 152.1 ± 7.154 mmHg in ecPOR-/-). In a rescue experiment using the NSAID naproxen, the increase in blood pressure induced by deletion of endothelial POR was abolished.
Conclusion: Collectively, in endothelial cells POR regulates eNOS activity and orchestrates the metabolic fate of arachidonic acid towards the vessel dilating EETs and away from deleterious prostanoids. In the absence of POR this endothelial regulation is compromised leading to vascular dysfunction.
Type 1 diabetes (T1D) is precipitated by the autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. Chemokines have been identified as major conductors of the islet infiltration by autoaggressive leukocytes, including antigen-presenting cells and islet autoantigen-specific T cells. We have previously generated a roadmap of the gene expression in the islet microenvironment during T1D in a mouse model and found that most of the chemokine axes are chronically upregulated during T1D. We focused our attention on CXCL10/CXCR3, CCL5/CCR5, CXCL16/CCR6, CX3CL1/CX3CR1, and XCL1/XCR1. First, we found that the absence of CCR6 and of CX3CR1 diminished T1D incidence in a mouse model for T1D. Further, the XCL1/XCR1 chemokine axis is of particular interest, since XCR1 is exclusively expressed on convention dendritic cells type 1 (cDC1) that excel by their high capacity for T cell activation. Here we demonstrate that cDC1 expressing XCR1 are present in and around the islets of patients with T1D and of islet-autoantibody positive individuals. Further, in an inducible mouse model for T1D, we show that XCL1 plays an important role in the attraction of highly potent dendritic cells expressing XCR1 to the islets. XCL1-deficient mice display a diminished infiltration of XCR1+ cDC1 and subsequently also a reduced magnitude and activity of islet autoantigen-specific T cells. XCR1-deficient mice display a reduced magnitude and activity of islet autoantigen-specific T cells. A 3D-visualization of the entire pancreas reveals that both XCL1-deficient mice and XCR1-deficient mice indeed maintain most of their functional islets after induction of the disease. Thus, the absence of XCL1 results in a profound decrease in T1D incidence. The XCR1-deficiency also reduces T1D incidence, even if in a less drastic way compared to XCL1-deficiency. An interference with the XCL1/XCR1 chemokine axis might constitute a novel target for the therapy for T1D.
The impact of the Covid-19 pandemic called for rapid responses in face of unprecedented challenges. In this context, earning more about the causative agent SARS-CoV-2 becomes imperative. Therefore, clinical virus isolates were studied with focus on infectivity, replication kinetic, and caspase activity.
Firstly, clinical specimens collected from patients were tested for infectivity in cell culture. Combined with polymerase chain reaction results, a formula predicting infectivity in cell culture based on abundance of viral RNA was developed. Additionally, analysis of different specimen types, sources, and material, elucidate the question of infectivity. Here, infectivity was demonstrated in specimens derived from different parts of the respiratory tract, including specimens collected from deceased persons. A protocol for virus isolation on human airway epithelium in air-liquid interface culture was established.
Secondly, replication kinetics of 20 clinical isolates were compared, including a subset of seven sequenced isolates. All isolates replicated in the colon epithelial cell culture model. Within the subset, differences between isolates carrying the D614G amino acid exchange and with original spike protein were observed.
Lastly, elevated caspase activity was demonstrated in two cell culture models including human airway epithelium in air-liquid interface culture.
Subsequently, caspase inhibition by small-molecule compound Emricasan and its effects on the cytopathic effect observed in cell culture were studied. Here, increased cell survival in a colon epithelial cell line was shown with unimpaired virus replication. Elevated caspase activity was identified as early marker of infection and validated by testing across 20 clinical virus isolates.
This study offers information on infectivity that can help shape the understanding of transmission risk. As such, parts of the data collected here were used for validation of rapid antigen tests. The insights gained by studying caspase activity contributed in part to the development of a drug screening method by Bojkova et al.,41 thus aiding routine laboratory workflow. It was demonstrated that Emricasan exhibits no antiviral effect, while the finding of increased cell survival in cell culture could give rise to further research on prevention of tissue damage.
IL-38 is the latest discovered cytokine of the IL-1 family and has been added to the IL-36 subfamily. Since its discovery in 2001, increasing evidence suggests predominantly anti-inflammatory properties of IL-38, which are most likely exerted through three potential receptors, the IL-1 Receptor 1 (IL-1R1), IL-36 Receptor (IL-36R) and the IL-1 Receptor Accessory Protein Like 1 (IL-1RAPL1). However, to this date detailed knowledge of IL-38 functioning remains to be examined. Importantly, how IL-38 is processed, secreted from cells and the exact mechanisms of target receptor binding and intracellular signaling are not fully understood. Further, IL-38 has been associated with regulatory functions in autoimmune diseases like systemic lupus erythematosus (SLE) and psoriasis. At the same time however, connections between B cells as indispensable part of immunity and IL-38 remain rare.
In this study we examined the influence of IL-38 in peripheral human blood B cells differentiating into antibody secreting cells using a three-step in vitro differentiation process. We first show that all potential IL-38 binding receptors are present on peripheral blood B cells on a gene expression level and remain detectable throughout B cell differentiation. Next, while B cells treated with exogenous IL-38 depict no differences in early B cell activation markers, the process of B cell differentiation revealed significant alterations in B cell phenotype created by IL-38 treatment. Predominantly on day 7 of the differentiation process, IL-38 treated B cells showed significantly reduced CD38 expression which depicts an important step in development towards plasma cells. We hypothesize that IL-38 acts antagonistically on the IL-1R1 pathway reducing Nuclear factor kappa B (NFκB) expression and consequently decreasing CD38 expression. Further IL-38 reduced early antibody production while increasing IgM secretion at the end stages of differentiation. Next, we repeated the differentiation assays under the influence of additional IL-21 stimulation to further enhance plasma cell development. In these experiments, the impact of IL-38 on B cell differentiation and immunoglobulin production were reduced, indicating a comparatively moderate relevance of IL-38 for B cell differentiation. We then examined how proliferation and cell death were impacted by exogenous IL-38 during B cell differentiation. IL-38 treatment alone significantly reduced B cell survival which was further augmented by IL-21 stimulation. We conclude that IL-38 and IL-21 act synergistically in promoting B cell apoptosis, also depicting an anti-inflammatory property of IL-38. Finally, using a siRNA we successfully performed an IL-38 knockdown experiment of human blood B cells reducing IL-38 expression to 44% measured on day 4 of B cell differentiation. In these experiments we observed reversed tendencies of CD38 expression compared to exogenous IL-38 treatment. Here, IL-38 knockdown cells showed increased CD38 expression indicating endogenous regulatory properties of IL-38 in B cell differentiation.
Our project, for the first time proves direct effects of IL-38 on human B cells. The results support previous research of IL-38 to act anti-inflammatory as it seems to modulate B cell differentiation, survival, and immunoglobulin production in a down-regulatory manner. These findings pave way for more detailed research on the connection between B cell homoeostasis and IL-38 function.
Abdominal aortic aneurysm (AAA) is the most common type of aortic aneurysm, which is defined as a dilation of the abdominal aorta over 3.0 cm or more. Surgical repair is the golden standard for the treatment of AAA, in which open surgical repair (OSR) and endovascular aneurysm repair (EVAR) are the main approaches. Technically speaking, the lesion segment of aueurysm is completely replaced by a graft during OSR, while in EVAR, the lesion is insulated by a stentgraft. EVAR is a less invasive treatment than OSR and shows a lower early mortality rate, although the long-term advantages of EVAR over OSR remain inconclusive.
Endoleak, especially the type II endoleak (T2EL), is a common complication after EVAR. According to research, 16-28% of the patients develop a T2EL after EVAR, and it accounts for nearly three in four of all types of endoleaks. Around 30-50% of the T2EL resolved spontaneously during the follow-up, however, it still causes a secondary intervention in many patients. Therefore, it is critical to monitor endoleaks after repair.
Patent aortic branches in the stent-overlapped area and vasa vasorum have been identified as potential sources of blood flow in T2EL. However, the mechanisms of biological changes or remodeling of the aneurysm sac after the repair are still not clear, but they have been considered to play an important role in the development of endoleaks. Unfortunately, it is impossible to obtain a tissue sample of the aortic wall in patients who underwent EVAR.
MicroRNAs (miRNAs) are a class of small single-stranded non-coding RNAs that inhibit the expression of target message RNA (mRNA). miR-29b/29c, miR-155, and miR-15a are miRNAs associated with regulating extracellular matrix (ECM) components, inflammation, and proliferation, respectively. All four miRNAs have been identified as biomarkers of AAA, not only in aneurysm tissue but also extracellular as circulating miRNAs. However, it is still unknown whether they can reflect the biological changes after AAA repair. Thus, we conducted a prospective study to investigate the changes in expression of circulating miR-29b, miR-29c, miR-155, and miR-15a before (T0), 3 days (T1), and 3 months (T2) after AAA repair.
A total of 39 patients were recruited for this study, 17 of whom were repaired by OSR and 22 of whom were repaired by EVAR. Four patients failed the T2 follow-up due to the Covid-19 pandemic. No significant changes were found in the expression of miR-29b, miR-29c, miR-155, and miR-15a. There were also no obvious differences between OSR and EVAR. However, the T1 expression of miR-15a was significantly lower in patients without endoleak after EVAR than in those who developed endoleak after EVAR and those who were repaired by OSR. Unfortunately, these differences did not persist to the T2 follow-up, and no other differences were found among these patients.
In summary, miR-15a is a miRNA that significantly changes in AAA patients. This study demonstrates that the expression of circulating miR-15a is lower in patients without endoleak three days after EVAR, compared to those who had endoleak after EVAR and those who underwent OSR. The results suggest that miR-15a might be involved in the early aortic remodeling after EVAR as an indicator of endoleak.
Polyunsaturated fatty acids (PUFAs) play essential roles in mediating inflammation and its resolution. PUFA metabolites generated by the cytochrome P450 (CYP) - soluble epoxide hydrolase (sEH) axis are known to regulate macrophage activation/polarization but little is known about their role in the resolution of inflammation. Monocytes were isolated from murine bone marrow or human peripheral blood and differentiated to naïve macrophages (M0). Thereafter cells were polarized using LPS and IFNγ (M1), IL-4 (M2a), or TGFβ1 (M2c). Gene expression was analyzed by RNA sequencing, RT-qPCR and Western blotting. Phagocytosis of zymosan and oxo-LDL were also assessed in vitro. Zymosan-induced peritonitis combined with immune cell profiling was used to evaluate the resolution of inflammation in vivo. The expression of sEH was comparable in M0, M1 and M2a macrophages but markedly elevated in M2c polarized cells. The increase in sEH expression elicited by TGFβ relied on the TGFβ receptor ALK5 and the phosphorylation of SMAD2, which was able to bind to the sEH promoter. In macrophages lacking sEH, M2c polarization was incomplete and characterized by lower levels of pro-resolving phagocytosis associated receptors (Tlr2 and Mrc1), as well as higher levels of the pro-inflammatory markers; Nlrp3, IL-1β and TNFα. Fitting with the failure to upregulate phagocytosis associated receptors, the uptake of zymosan and ox-LDL was less efficient in M2c macrophages from sEH-/- mice. The latter animals also demonstrated a retarded resolution of inflammation (zymosan-induced peritonitis) in vivo with fewer resident macrophages and recruited macrophages. PUFA profile analysis indicated decreased sEH substrates e.g., 11, 12-EET, as well as increased sEH products e.g., 11, 12-DHET, indicating an increased sEH activity in M2c macrophages. Taken together, our data indicates that sEH expression is required for the effective M2c polarization of macrophages and thus the resolution of inflammation.
Inflammation is a crucial host defense mechanism activated in response to injury or infection. Its primary goal is to eliminate the source of the disturbance, repair the damaged tissue, and restore homeostasis. Inflammatory processes can be recognized through increased blood flow, higher vascular permeability, and the recruitment of leukocytes and plasma proteins to the tissue. A pathogen-induced inflammation triggers various pro- and anti-inflammatory processes. Local tissue cells and Toll-like receptors call upon innate immune cells like neutrophils, dendritic cells (DCs), and monocytes to respond to the intruder. They move across the endothelium and respond to local signals by releasing mediators or cytotoxic compounds, phagocytosing, or polarizing. To study local pathogen-induced inflammation, a zymosan-induced inflammation model was used in the hind paws of mice, which caused a Toll-like receptor 2 mediated inflammation. Multi-Epitope-Ligand-Cartography (MELC) was used for multiple sequential immunohistochemistry with 40 different antibodies on the same tissue. Bioinformatic analysis and graphical representation revealed a specific inflammatory architecture consisting of three major areas based on macrophage polarization and their cellular neighborhoods: a core region containing the pathogen, a pro-inflammatory region containing M1-like macrophages, and a region containing anti-inflammatory cells. This discovery highlights the coexistence of pro- and antiinflammatory processes during an ongoing inflammation and challenges the concept of a gradual temporal transition from pro- to anti-inflammation. Flow cytometry of the whole paw was performed to support and refine the MELC results. Eosinophils were used as a specific immune cell population to investigate their role in the inflammatory structure. They were found to be present in all three inflammatory regions, adapting their cytokine profile according to their localization. Depleting eosinophils reduced Interleukin 4 (IL-4)- levels, increased edema formation, and mechanical and thermal hypersensitivities during inflammation resolution. In the absence of eosinophils, pro- and anti-inflammatory region could not be determined in the inflammatory architecture, neutrophil numbers increased, and efferocytosis and M2-macrophage polarization were reduced. IL-4 administration restored these regions, normalized neutrophil numbers, efferocytosis, M2-macrophage polarization, and resolution of zymosan-induced hypersensitivity. The results show that eosinophils expressing IL-4 support the resolution of inflammation by enabling the development of an anti-inflammatory framework that encloses pro-inflammatory regions.
Pericytes are capillary-associated mural cells involved in the maintenance and the stability of the vascular network. This thesis aims to investigate the role of pericytes in the heart in the context of ageing and disease. We highlight the malignant effects of the remodelling in the heart and stress the focus on the role of cardiac pericytes in this context. We show that ageing reduces pericyte coverage and that myocardial infarction (MI) causes an activation of these cells. Single-nuclei and single-cell RNA sequencing analysis of murine hearts further revealed that the expression of the Regulator of G-protein signalling 5 (Rgs5) is reduced in cardiac pericytes both in ageing and transiently at day 1 and day 3 after MI. The loss of RGS5 in pericytes drives an entropic state of these mural cells characterized by morphological changes, excessive extracellular deposition and enhanced Gaq mediated GPCR signalling. The deletion of RGS5 in pericytes causes cardiac systolic dysfunction, induces myocardial fibrosis, and drives the activation of cardiac fibroblasts in a TGFb-dependent manner. In conclusion, our results describe the importance of pericytes maintaining cardiac homeostasis, identify RGS5 as a key regulator of this process and propose pericytes as crucial mediators of cardiac fibrosis and possible therapeutic targets to prevent cardiovascular disease.
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with an onset in early development. ASD has varying degrees of severity and thus affects people differently throughout their lives. Early diagnosis of ASD is essential to provide children with individually-tailored support.8 Eye-tracking may contribute to an earlier diagnosis: Several studies showed differences in eye movements between people with autism spectrum disorder (ASD) and typically developing controls (TD). Different eye movements may contribute to different visual perception that perpetuates to problems in attention, communication and social interaction.
Eye movements are divided into: (1) Fixations (2) Saccades (fast and short eye movements) and (3) Smooth Pursuit Eye Movements (SPEM). SPEM follow the target in a continuous manner. The latter are the subject of the present thesis. SPEM consist of two phases: the open loop phase (= phase of initiation, first 50- 100ms) and the closed loop phase (= phase of maintenance, after about 100ms). SPEM are usually measured by a gain index. It is defined as the ratio of smooth pursuit velocity and visual target velocity and ideally equals to 1.2
In young children, corneal-reflection (CR) eye-tracking is usually applied to quantify eye movement. It allows precise measurements without the use of potentially intrusive devices.
Studies in ASD reported deficits in open loop and closed loop pursuit in children and adults with a mean age of 19.32 (TD) and 20.04 (ASD) years. However, SPEM in preschoolers with ASD remain understudied, although this developmental phase is crucial to the development of non-social and social attentional abilities.
In the present study 66 toddlers and preschoolers (18 to 72 months; ASD: n = 33, TD: n = 33) with matched cognitive abilities and sex were assessed. The main objective was to compare the gain index (Smooth Pursuit Gain = SPG). SPEM were compared between groups with gain index as a dependent measure. We hypothesized that participants with ASD show lower average gain compared to the control group.
We could show a significant group influence on the gain when considering interactions between target velocity and group (p = 0.041). The TD group showed a greater dependence on the increasing object speed than the ASD group with a trend of -0.30 ± 0.11 in the TD group and a trend of -0.13 ± 0.12 in the ASD group. Across groups, the gain decreased with increasing target velocity and dropped faster in vertical than in horizontal trials. Additionally, participants showed a lower SPG in vertical sequences than in horizontal sequences. This supports the general validity of the measure.
Toddlers and preschoolers represent a group that has been subject of little research to date. In addition, there has been only a limited number of studies analyzing SPEM in ASD. To check for a possible group difference without interactions a study with a larger sample size at fixed target velocity and target direction should follow.