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Identifizierung von Kollagenosepatienten mit unterschiedlicher TLR7 und TLR9 abhängiger Interferonproduktion durch plasmazytoide dendritische Zellen: Entwicklung einer Methode
Full text linkKollagenosen umfassen eine heterogene Gruppe komplexer, chronischer und lebensbedrohlicher Autoimmunerkrankungen unbekannter Ätiologie, die zu multiplen Organpathologien wie einer Nierenbeteiligung führen können. Dabei wird deren antinukleäre Autoantikörperproduktion insbesondere mit den NA-TLRs TLR7 und TLR9 assoziiert, aus der eine erhöhte IFN-α Produktion resultiert. Tierexperimentelle Versuche zeigen, dass die Rolle von TLR7 in der SLE-Pathogenese verstärkt wird, wenn die regulatorische Rolle von TLR9 fehlt. In dieser Arbeit wurde in einem translationalem Ansatz eine kleine Kollagenosekohorte (n = 14) auf TLR7 und/oder TLR9 Pathwaydefekte anhand einer differenziell veränderten Zytokinantwort untersucht. In diesem Zuge wurde ein Antikörper-Panel etabliert, welches erlaubt pDCs durchflusszytometrisch exakt zu detektieren und gleichzeitig deren intrazelluläre Zytokinproduktion (IFN-α, TNF-α) nach dosisabhängiger Stimulation mit TLR7 und TLR9 Agonisten auf Einzelzellebene zu messen. Nach TLR9 Stimulation konnte eine signifikante Reduktion des prozentualen Anteils der IFN-α+ produzierenden pDC im Vergleich zur gesunden Kohorte festgestellt werden. Gleichzeitig war die Fähigkeit der pDCs auf Einzelzellebene IFN-α zu produzieren nach TLR9 Stimulation signifikant gesteigert. Dieses Ergebnis führte erstmalig zur Neubewertung des Begriffes „IFN-α produzierenden Kapazität“, in der neben den prozentualen Anteil der IFN-α+ pDCs auch der MFI der IFN-α+ pDCs, als semiquantitatives Maß für die (intrazelluläre) Zytokinexpression pro Zelle, mitbewertet wird. Hinsichtlich der TNF-α produzierenden Kapazität der pDCs ergaben sich nach TLR7 bzw. TLR9 Stimulation keine signifikanten Änderungen. Außerdem ließen sich anhand der erhobenen Daten die pDCs definiert nach ihrer TNF-α und IFN-α Produktion in vier funktionelle Subgruppen unterteilen: die „non producer“ IFN-α-TNF-α-, die einfachpositiven IFN-α+TNF-α- und IFN-α-TNF-α+ und die doppelpositiven IFN-α+TNF-α+. Hierbei zeigte sich die Subgruppe der einfachpositiven IFN-α+TNF-α- pDCs der Kollagenosepatienten nach TLR9 Stimulation im Vergleich zur gesunden Kohorte signifikant reduziert. Bei individueller Betrachtung der dosisabhängigen TLR7 bzw. TLR9 Antwort konnten keine größeren TLR7 und TLR9 Pathwaydefekte bei den Patienten festgestellt werden. Des Weiteren war keine Korrelation zwischen dem Anti-dsDNA Antikörpertiter der Patienten und der TLR7 bzw. TLR9 vermittelte Häufigkeit der IFN-α+ pDCs festzustellen.
Zusammenfassend konnte in dieser Arbeit eine Methode zur durchflusszytometrisch Untersuchung von pDCs und deren intrazelluläre Zytokinproduktion (IFN-α, TNF-α) auf Einzelzellebene etabliert werden. Es wurde erstmalig bei der Bewertung der IFN-α bzw. TNF-α produzierenden Kapazität der pDCs neben dem prozentual positiven Anteil auch der Aspekt der mittleren Zytokinproduktion auf Einzelzellebene (mit Hilfe des MFI) mitberücksichtigt. Hinzu erfolgte eine Einteilung der pDCs definiert nach ihrer TNF-α und IFN-α Produktion in vier funktionelle Subgruppen. An einer größeren Kollagenosepatientenkohorte sollte eine Fortsetzung der Untersuchungen, die auf die in der vorgelegten Arbeit etablierten Methode aufbauen, erfolgen. Dadurch können einzelne Patienten mit TLR7 und/oder TLR9 Pathwaydefekte identifiziert werden, bei der man mit Hilfe genetischer Testungen mögliche aberrante Transkripte oder Pathwaydefekte aufdecken könnte. Dies diene langfristig zur Entwicklung individualisierter Therapieansätze für eine zielgerichtete Therapie gegen Kollagenosen beizutragen
Doctor’s Orders—Why Radiologists Should Consider Adjusting Commercial Machine Learning Applications in Chest Radiography to Fit Their Specific Needs
Full text linkThis retrospective study evaluated a commercial deep learning (DL) software for chest radiographs and explored its performance in different scenarios. A total of 477 patients (284 male, 193 female, mean age 61.4 (44.7–78.1) years) were included. For the reference standard, two radiologists performed independent readings on seven diseases, thus reporting 226 findings in 167 patients. An autonomous DL reading was performed separately and evaluated against the gold standard regarding accuracy, sensitivity and specificity using ROC analysis. The overall average AUC was 0.84 (95%-CI 0.76–0.92) with an optimized DL sensitivity of 85% and specificity of 75.4%. The best results were seen in pleural effusion with an AUC of 0.92 (0.885–0.955) and sensitivity and specificity of each 86.4%. The data also showed a significant influence of sex, age, and comorbidity on the level of agreement between gold standard and DL reading. About 40% of cases could be ruled out correctly when screening for only one specific disease with a sensitivity above 95% in the exploratory analysis. For the combined reading of all abnormalities at once, only marginal workload reduction could be achieved due to insufficient specificity. DL applications like this one bear the prospect of autonomous comprehensive reporting on chest radiographs but for now require human supervision. Radiologists need to consider possible bias in certain patient groups, e.g., elderly and women. By adjusting their threshold values, commercial DL applications could already be deployed for a variety of tasks, e.g., ruling out certain conditions in screening scenarios and offering high potential for workload reduction
Age- and Sex-Specific Reference Values for Renal Volume and Association with Risk Factors for Chronic Kidney Disease in a General Population—An MRI-Based Study
Full text linkBackground: Renal volume (RV) is associated with renal function and with a variety of cardiovascular risk factors (CVRFs). We analysed RV using magnetic resonance imaging (MRI) in a large population-based study (Study of Health in Pomerania; SHIP-TREND) to find sex- and age-specific reference values for RV and to test the influence of several markers on RV. The main objective is to describe reference values for RV in people from the general population without kidney disease. Methods: 1815 participants without kidney disease (930 women) aged 21–81 years were included in our study. Right and left RV with and without body surface area (BSA) indexation were compared among three age groups (22–39 years, 40–59 years, 60–81 years) by median and interquartile range and tested separately in women and men. Results: The estimated glomerular filtration rate (eGFR), serum uric acid, and right and left RV were higher in men compared to women (all p < 0.001). Left kidneys were larger than right kidneys (both sexes). With age, RV showed a continuously decreasing trend in women and an upside-down U-shaped relation in men. In multivariable linear regression models, current smoking (β = 14.96, 95% CI 12.12; 17.79), BSA (β = 97.66, 95% CI 90.4; 104.93), diastolic blood pressure (β = 0.17, 95% CI 0.01; 0.32), and eGFR (β = 0.57, 95% CI 0.50; 0.65) were positively associated with both left and right RV, whereas uric acid (β = −0.03, 95% CI −0.05; −0.01) showed an inverse association with RV. Interestingly, the same eGFR correlated with higher RV in men compared to women. Conclusion: Reference values for RV are different for age groups and sex. For any given age, female kidneys are smaller than male kidneys. RV associates positively with eGFR, but for any chosen eGFR, renal volume in females is lower compared to males. RV decreases with age, but in men showed a U-shaped correlation. This may reflect hyperfiltration and glomerular hypertrophy associated with the presence of CVRF in middle-aged males
Dynamics and Coordination of a P2N2 Ligand – from Twisted Conformation to Chelation
Full text linkBased on their general spacial flexibility, their Lewis and Brønsted basicity, and ability to mimic second sphere effects the 1,5‐diaza‐3,7‐diphosphacyclooctane ligand family and their complexes have regained substantial scientific interest. It was now possible to structurally analyze a recently reported member of this family with p‐tolyl and t‐butyl substituents on P and N, respectively, (P2p‐tolN2tBu). Notably, the ligand crystallizes with a ‘twisted’ backbone. This compound is the very first of its kind to have been unambiguously characterized with regard to its chemical and molecular structure as being in this conformation. A temperature‐dependent NMR study provides insight into the molecular dynamics of two isomers in solution, which are most likely also both twisted, as judged by the observed limited reactivity. Despite the in principle unfavorable conformation of the free ligand, it was successfully chelated to tungsten and molybdenum centers in mononuclear carbonyl complexes. The ligand, a derivative thereof and four new complexes were comprehensively characterized and analyzed in comparison. This includes single crystal XRD molecular structures of P2p‐tolN2tBu and all four complexes. P2p‐tolN2tBu, regardless of its twisted conformation, is able to coordinate to metal centers given that enough energy (heat) for a conformational change is provided
Zerebrale Metastasierung beim Malignen Melanom; Systematische Untersuchung der Krankheitsverläufe von Patienten des Greifswalder Hauttumorzentrums
Full text linkZusammenfassung
Bei dieser Arbeit handelt es sich um eine retrospektive systematische Untersuchung einer Kohorte von 45 Patienten, welche auf Grund der zerebralen Metastasierung eines Malignen Melanoms am Hauttumorzentrum der Universitätsmedizin Greifwald behandelt wurden.
Der Zeitraum der Erfassung belief sich basierend auf dem progressiven Therapiewandel auf den Zeitraum zwischen 2011 und 2021.
Der resultierende Datensatz wurde in ACCESS Datenbanken erfasst und anschließend mit Hilfe von Kaplan-Meier Analysen auf das Überleben unter Berücksichtigung von verschiedenen Einflussfaktoren statistisch ausgewertet.
Das Patientenkollektiv setzte sich aus 57,78 % männlichen und 42,22 % weiblichen Patienten zusammen.
Der Mutationsstatus lag bei 51,1 % BRAF-mutierten und 11,1 % NRAS-mutierten Patienten. Zum Zeitpunkt der Diagnose der zerebralen Metastasierung lag das mediane Alter bei 68 Jahren und das mittlere Überleben bei 107 Tagen, jedoch zeigte sich mit einem Verhältnis von 57 Tagen zu 215 Tagen ein signifikanter Überlebensvorteil des mittleren Überlebens gegenüber dem weiblichen Geschlecht (p=0,001).
Ein signifikantes Ergebnis zeigte sich ebenfalls in der medianen Überlebenszeit von Patienten mit einem normwertigen (211 Tage) und Patienten mit einem erhöhten S100B-Wert am Ende der Therapien (107 Tage) (p=0,007).
Therapeutisch konnte mit schwacher Signifikanz ein Überlebensvorteil des mit Checkpoint- Inhibitoren behandelten Kollektivs, gegenüber der nicht Checkpoint-therapierten Gruppe nachgewiesen werden (p=0,0645).
Unter den zielgerichteten Therapien tat sich die Behandlung mit Dabrafenib + Trametinib hervor, da sie einen besonderen Vorteil der mittleren Überlebenszeit darstellte.
Die stereotaktische Einzeitbestrahlung zeigte in dieser Untersuchung gegenüber der Ganzhirnbestrahlung ein verbessertes mittleres Überleben von 1,74 Monaten.
Einigen Symptomen, wie neurologische Ausfallerscheinungen oder Krampfanfällen konnte ein Überlebensvorteil nachgewiesen werden. Dies wird hier mit einer früheren Diagnose der Zerebralmetastasen und dem damit assoziierten frühzeitigeren Therapiebeginn erklärt.
Das mittleres Sterbealter des Kollektivs liegt bei 73,5 Jahren.
Alle verstorbenen Patienten sind den Folgen der zerebralen Metastasierung erlegen.
Insgesamt konnten viele Ergebnisse weiterer Untersuchungen bestätigt werden
Rational Engineering of Natural Ribozymes: From Canonical to Novel Functions
Full text linkThe objective of this thesis is to examine the evolution of ribozymes with novel functions through the application of rational engineering based on the remodelling of natural ribozymes. The development of these new functions may contribute to the concept of the RNA world. In order to demonstrate the potential of RNA as an early life form preceding the evolution of DNA and protein, it is essential that it exhibits a range of functions independently. The majority of these engineering efforts are based on hairpin ribozymes. The selection of hairpin ribozymes as the blueprint for this study was based on three key factors: their simple structural composition, sequence flexibility and the reversibility of the catalytic reaction they facilitate. These characteristics provide broad space and possibilities for design
Study on the ex vivo effects of neurotrophin-3 on human T cell and monocyte activation post stroke
Full text linkConsidering the great epidemiologic and economic significance of stroke-related
disability, it is of importance to develop treatment options which may restore
neurological deficits post stroke. As a cumulating body of evidence suggests, the
immune system plays a key role regarding damage and regeneration in the acute
and chronic phases post stroke. In this context, it has been hypothesized that
neurotrophin-3 (NT-3) can boost neurological recovery even months after brain
lesioning upon stimulation of the immune system e.g. with lipopolysaccharide (LPS).
The present study aimed to reveal the effects of NT-3 treatment on T cell and
monocyte activation using blood samples from controls and stroke patients on day 3
post stroke. Cells were incubated for 48 hours with T cell specific anti-CD3/CD28
stimulation with or without NT-3. Expression of activation markers CD25, CD69 and
HLA-DR by T cells and HLA-DR by monocytes as well as cytokine concentrations
were quantified by flow cytometry (FC). Besides, expression levels of high-affinity
NT-3 receptor TrkC in T cells and monocytes from young and elderly controls were
assessed by FC after stimulation with anti-CD3/CD28 or LPS with or without NT-3.
In general, T cells and monocytes in stroke patient samples were observed to
react more likely to NT-3 treatment by down-regulating activation marker expression
than cells from controls. Cytokine concentrations were decreased upon NT-3
treatment in both cohorts. The percentage of cells expressing TrkC was highest in
monocytes from elderly control samples, but less sensitive to stimuli and NT-3.
For T cells, TrkC expression was highest with stimulation only and tended to decline
upon NT-3 treatment, most pronounced so for samples from young controls.
In conclusion, NT-3 reduced cytokine and activation marker expression of
stimulated T cells and monocytes. Concerning the acute stroke phase, such a
suppressive effect may restrict inflammatory damage to penumbra tissue. In the
chronic phase, the development of autoimmunity against brain antigens may be
mitigated, improving long-term outcome. In the future, TrkC expression of T cells
derived from aged stroke patients months after stroke should be quantified,
especially upon stimulation with LPS, to assess NT-3 susceptibility
Ultraviolet laser induced periodic surface structures positively influence osteogenic activity on titanium alloys
Full text linkBackground/Objective
Endoprostheses might fail due to complications such as implant loosening or periprosthetic infections. The surface topography of implant materials is known to influence osseointegration and attachment of pathogenic bacteria. Laser-Induced Periodic Surface Structures (LIPSS) can improve the surface topography of orthopedic implant materials. In this preclinical in vitro study, laser pulses with a wavelength in the ultraviolet (UV) spectrum were applied for the generation of LIPSS to positively influence formation of extracellular matrix by primary human Osteoblasts (hOBs) and to reduce microbial biofilm formation in vitro .
Methods
Laser machining was employed for generating UV-LIPSS on sample disks made of Ti6Al4V and Ti6Al7Nb alloys. Sample disks with polished surfaces were used as controls. Scanning electron microscopy was used for visualization of surface topography and adherent cells. Metal ion release and cellular metal levels were investigated by inductively coupled plasma mass spectrometry. Cell culture of hOBs on sample disks with and without UV-LIPSS surface treatments was performed. Cells were investigated for their viability, proliferation, osteogenic function and cytokine release. Biofilm formation was facilitated by seeding Staphylococcus aureus on sample disks and quantified by wheat germ agglutinin (WGA) staining.
Results
UV-LIPSS modification results in topographies with a periodicity of 223 nm ≤ λ ≤ 278 nm. The release of metal ions was found increased for UV-LIPSS on Ti6Al4V and decreased for UV-LIPSS on Ti6Al7Nb, while cellular metal levels remain unaffected. Cellular adherence was decreased for hOBs on UV-LIPSS Ti6Al4V when compared to controls while proliferation rate was unaffected. Metabolic activity was lower on UV-LIPSS Ti6Al7Nb when compared to the control. Alkaline phosphatase activity was upregulated for hOBs grown on UV-LIPSS on both alloys. Less pro-inflammatory cytokines were released for cells grown on UV-LIPSS Ti6Al7Nb when compared to polished surfaces. WGA signals were significantly lower on UV-LIPSS Ti6Al7Nb indicating reduced formation of a S. aureus biofilm.
Conclusion
Our results suggest that UV-LIPSS texturing of Ti6Al7Nb positively influence bone forming function and cytokine secretion profile of hOBs in vitro . In addition, our results indicate diminished biofilm formation on UV-LIPSS treated Ti6Al7Nb surfaces. These effects might prove beneficial in the context of long-term arthroplasty outcomes
Management of Bilateral Congenital and Juvenile Cataracts in a Low-Income Country: Patient Identification, Treatment Outcomes, and Follow Up
Full text linkBackground: Childhood blindness remains a neglected issue in eye care within low-income countries, with congenital and juvenile cataracts being the most common treatable causes. This study aims to investigate the factors influencing the management of congenital and juvenile bilateral cataracts, provide data on general outcomes and postoperative findings, and discuss treatment in a low-income country context. Methods: In this prospective study, data from clinical care were continuously entered into a database, and one follow-up examination of a cohort of 91 patients with congenital and juvenile bilateral cataracts in Kinshasa, Democratic Republic of the Congo, was conducted. Multiple factors that affect the first clinical presentation, the clinical management, and outcome were investigated. Results: Most patients (88.5%) who received medical treatment were identified by cataract finders. A patient’s presentation time was independent of the parent’s education, social status, income, and sex of the child. The median age at first presentation was 5.8 years (ranging from 0 days to 17.3 years). The best visual acuity outcomes were achieved by patients who underwent surgery during early childhood. According to WHO grades and on an eye level basis, 51.1% (93 out of 182 eyes) showed improvement, while 34.6% (63 eyes) had unchanged results post-surgery. Among the cases without improvement, 76.2% were blind prior to surgery. Postoperative findings were observed in 56% of the patients, with secondary cataracts being the most common, affecting 26.37% of the operated eyes. Conclusions: Optimal management of childhood cataracts involves early identification, efficient hospital infrastructure, high-quality medical care, and long-term follow up. In sub-Saharan Africa, this approach must be adapted to the context of available resources, which differs significantly from those in high- and middle-income countries
More I-talk in student teachers’ written reflections indicates higher stress during VR teaching
Full text linkVideo-based reflection on one's own teaching represents a crucial tool in teacher education. When student teachers reflect on negative classroom events, it elicits “self-focused attention,” which has been associated with more intense negative emotionality. Self-focused attention can be quantitatively captured using first-person singular pronouns (“I,” “me,” “my”) in written reflections by, for instance, student teachers. What is unclear is whether student teachers' use of these first-person singular pronouns in their written reflections is linked to and predicts their negative affective experiences during teaching. For the present study, a fully immersive virtual reality (VR) classroom was implemented in which student teachers taught a lesson, provided written reflections on their teaching, and then taught a second lesson. We measured N = 59 student teachers' self-reported stress and heartrate responses while teaching in the VR classroom and determined the percentage of first-person singular pronouns in their written reflections. Firstly, our results showed that the use of first-person singular pronouns provides incremental information on manual ratings of student teachers' foci in their written reflections. Secondly, student teachers' heartrates during instruction—a measure of physiological stress—were associated with the use of first-person singular pronouns in subsequent written reflections. Thirdly, the use of first-person singular pronouns predicted the increase in physiological stress from the first to the second round of VR teaching. We discuss implications for automated feedback and for designing reflective tasks