Incidence of and risk factors for vaginal cuff dehiscence following total laparoscopic hysterectomy: a monocentric hospital analysis

Purpose Vaginal cuff dehiscence (VCD) is one of the major surgical complications following hysterectomy with data on incidence rates varying largely and studies assessing risk factors being sparse with contradictive results. The aim of this study was to assess the incidence rate of and risk factors for VCD in a homogenous cohort of women treated for benign uterine pathologies via total laparoscopic hysterectomy (TLH) with standardized follow-up. Methods All patients undergoing TLH at the Department of Gynecology and Obstetrics, Saarland University Hospital between November 2010 and February 2019 were retrospectively identified from a prospectively maintained service database. Results VCD occurred in 18 (2.9%) of 617 patients included. In univariate and multivariate analyses, a lower level of surgeon laparoscopic expertise (odds ratio 3.19, 95% confidence interval (CI) 1.0–9.38; p = 0.03) and lower weight of removed uterus (odds ratio 0.99, 95% CI 0.98–0.99; p = 0.02) were associated positively with the risk of VCD. Conclusion In this homogenous cohort undergoing TLH, laparoscopic expertise and uterine weight influenced the risk of postoperative VCD. These findings might help to further reduce the rate of this complication

MRI-detected brain lesions in AF patients without further stroke risk factors undergoing ablation - a retrospective analysis of prospective studies

Abstract Background Atrial fibrillation (AF) without other stroke risk factors is assumed to have a low annual stroke risk comparable to patients without AF. Therefore, current clinical guidelines do not recommend oral anticoagulation for stroke prevention of AF in patients without stroke risk factors. We analyzed brain magnetic resonance imaging (MRI) imaging to estimate the rate of clinically inapparent (“silent”) ischemic brain lesions in these patients. Methods We pooled individual patient-level data from three prospective studies comprising stroke-free patients with symptomatic AF. All study patients underwent brain MRI within 24–48 h before planned left atrial catheter ablation. MRIs were analyzed by a neuroradiologist blinded to clinical data. Results In total, 175 patients (median age 60 (IQR 54–67) years, 32% female, median CHA2DS2-VASc = 1 (IQR 0–2), 33% persistent AF) were included. In AF patients without or with at least one stroke risk factor, at least one silent ischemic brain lesion was observed in 4 (8%) out of 48 and 10 (8%) out of 127 patients, respectively (p > 0.99). Presence of silent ischemic brain lesions was related to age (p = 0.03) but not to AF pattern (p = 0.77). At least one cerebral microbleed was detected in 5 (13%) out of 30 AF patients without stroke risk factors and 25 (25%) out of 108 AF patients with stroke risk factors (p = 0.2). Presence of cerebral microbleeds was related to male sex (p = 0.04) or peripheral artery occlusive disease (p = 0.03). Conclusion In patients with symptomatic AF scheduled for ablation, brain MRI detected silent ischemic brain lesions in approximately one in 12 patients, and microbleeds in one in 5 patients. The prevalence of silent ischemic brain lesions did not differ in AF patients with or without further stroke risk factors

Gene expression analyses of murine bone marrow stromal cells and their neuronal differentiation

Titelblatt Inhaltsverzeichnis, Abkürzungsverzeichnis Einleitung Material Methoden Ergebnisse Diskussion Zusammenfassung Abstract Anhang und LiteraturverzeichnisKnochenmarkstromazellen (KMSZ) wurden zum ersten Mal in den Siebziger Jahren durch ihre selektive Adhäsion an Kunststoffoberflächen von Zellkulturgefäßen gewonnen und als Kolonie-bildende Fibroblasten ("colony-forming-unit fibroblasts", CFU-F) beschrieben. Schon früh zeigte sich ihre Fähigkeit, in verschiedene mesenchymale Zelltypen wie Osteoblasten, Chondrozyten und Adipozyten zu differenzieren. KMSZ werden deshalb auch als mesenchymale Stammzellen bezeichnet. In den letzten Jahren zeigten verschiedene Publikationen die Differenzierung in vivo oder in vitro in weitere Zelltypen wie Myoblasten, Zellen des Lungenepithels, Herzmuskelzellen und neurale Phänotypen (Astrozyten und Neuron-ähnliche Zellen). Damit differenzieren KMSZ nicht nur in nahe verwandte Zelltypen, sondern sogar in Zellen, die unterschiedlichen Keimblättern zugerechnet werden. Die Fähigkeit von KMSZ, in vitro einen neuronalen Phänotyp zu entwickeln, wurde unter anderem in der Arbeit von Woodbury et al. (2000) gezeigt. Die Ursachen der Differenzierungsfähigkeit von KMSZ sind nicht bekannt. Weiterhin herrschte bis zum Beginn der vorliegenden Studie Unklarheit über den genauen (in vivo) Phänotyp von KMSZ. In der vorliegenden Arbeit sollte durch Genexpressionsanalysen mit Hilfe von cDNS-Mikroarrays ein molekulares Profil muriner KMSZ erstellt und ihre Differenzierung in Zellen mit einem neuronalen Phänotyp in einer Zeitreihenanalyse untersucht werden. Undifferenzierte KMSZ der Maus exprimieren in vitro Gene, die für unterschiedliche Zelltypen wie z.B. neurale Zellen, verschiedene Muskelzelltypen, Zellen des Bindegewebes sowie Gefäß-assoziierte Zellen charakteristisch sind. Literaturvergleiche ergaben eine große molekulare Ähnlichkeit mit Perizyten. Perizyten sind Zellen, die in der Basalmembran kleiner Gefäße lokalisiert sind. Auch morphologisch besteht in vitro eine große Ähnlichkeit zwischen Perizyten und KMSZ. Zur genaueren Untersuchung wurden Kryoschnitte des adulten Knochenmarks mit Antikörpern gegen verschiedene Proteine von in KMSZ exprimierten Genen markiert, u.a. Cadherin 13, Vimentin, Slug und VE-Cadherin. Alle färbten übereinstimmend Gefäß-assoziierte Zellen an und unterstützen damit die Vermutung, daß es sich bei KMSZ um Perizyten des Knochenmarks handelt. Die neuronale Differenzierung der KMSZ wurde zunächst durch Immunfärbungen neuraler Proteine (u.a. Neurofilament-M, Neuronen-spezifische Enolase, Nestin, RC2, GFAP, O4, GalC) validiert. Etwa 85% der kultivierten KMSZ synthetisierten nach zweitägiger Inkubation im Differenzierungsmedium NF-M, keine der kultivierten Zellen Marker für Astrozyten (GFAP) und Oligodendrozyten (O4, GalC). Während der neuronalen Differenzierung muriner KMSZ änderte sich das Expressionsmuster vieler Gene, die für Proteine z.B. des Zytoskeletts, der extrazellulären Matrix sowie Komponenten der Signaltransduktion kodieren. Eine Funktion in der neuronalen Differenzierung wurde für verschiedene der festgestellten Gene bereits beschrieben. Aufgrund der differentiellen Expression des Transkriptionsfaktors Slug, der unter anderem entscheidend für die Epithelial-mesenchymale Transition während der Entstehung von Neuralleistenzellen ist, wurde die Expression weiterer Neuralleisten- spezifischer Gene, Noelin, Twist und Snail, untersucht. Diese sind wie Slug bereits in undifferenzierten KMSZ exprimiert. Auch die Expressionsänderungen der Gene von Sox-8, -9 und -10, Wnt-1 und -3a sowie Pax-3 und -6 im Laufe der Differenzierung deuten auf Ähnlichkeiten der neuronalen Differenzierung adulter KMSZ mit Regulationsmechanismen der frühen neuronalen Entwicklung hin, genauer der Entstehung und Spezifizierung der Neuralleistenzellen. Diese Ähnlichkeiten werfen die Frage auf, ob KMSZ während ihrer neuronalen Differenzierung ein molekulares Programm aktivieren, welches ursprüngliche Entwicklungsschritte der neuronalen Entwicklung rekapituliert, oder ob KMSZ vielleicht selbst von Neuralleistenzellen abstammen. Mit einer möglichen neuroektodermalen Abstammung von KMSZ ließe sich ihr Differenzierungspotential erklären und es ergäben sich völlig neue Perspektiven, sie weiter entwicklungsbiologisch zu untersuchen.Bone marrow stromal cells (BMSC) were first identified by their adherence to plastic tissue culture dishes and termed "colony forming-unit fibroblasts" (CFU-Fs). These had the ability to differentiate into different mesenchymal cells like osteoblasts, chondrocytes and adipocytes. For that reason, BMSC are also called mesenchymal stem cells (MSCs). In recent years several publications showed the differentiation of BMSC in vitro or in vivo into myoblasts, epithelial cells, cardiac muscle cells and neural phenotypes, i.e. BMSC differentiate even into cells of different germ layers. The fascinating capacity of BMSC to develop a neuronal phenotype in vitro was for example shown by Woodbury and colleagues (2000). The differentiation of BMSC towards a neuronal phenotype raises the question of the mechanisms underlying these processes. Furthermore, the exact (in vivo) identity of BMSC had not been known in the beginning of this study. Therefore, gene expression analyses of murine BMSC using cDNA-microarrays were performed to establish a molecular profile of these cells and their neuronal differentiation. Undifferentiated BMSC express genes that code for proteins of different cells like neural cells, different muscle cells, mesenchymal cell types and vascular-associated cells. Literature-analyses of the expressed genes showed a high similarity to pericytes. Pericytes are localized in the basement membrane of small vessels like capillaries. Also the morphology of BMSC and pericytes in vitro is very similar. To confirm a vascular-associated phenotype of BMSC in vivo, slices of adult murine bone marrow were marked for proteins that are expressed by BMSC in vitro, e.g. Cadherin 13, Vimentin, Slug and VE-Cadherin. All antibodies stained blood vessel-associated cells and therefore support the proposed pericytic nature of BMSC in vivo. The neuronal differentiation of murine BMSC was validated with several antibodies against neural proteins (e.g. neurofilament-M, neuron-specific enolase, nestin, RC2, GFAP, O4). About 85% of the cells were positive for the neuronal marker NF-M after 2 days of differentiation, none of the cells expressed markers for astrocytes (GFAP) or oligodendrocytes (O4, GalC). The time-course analysis of the neuronal differentiation of murine BMSC with cDNA-microarrays revealed gene expression changes of genes that code for proteins of for example the cytoskeleton, the extracellular matrix or components of signal transduction pathways. Many of those were already correlated to the process of neuronal differentiation. Because of the differentially expressed gene of the transcription factor Slug, that is responsible for epithelial-mesenchymal transition (EMTs) in the development of neural crest cells, the expression of other neural crest- specific genes was analysed (Twist, Snail and Noelin). These genes are also already expressed in undifferentiated BMSC. The differential expression of Sox-8, -9 and -10, Wnt-1 and -3a, as well as Pax-3 and -6 in course of the differentiation reveals similarities of the neuronal differentiation of BMSC with processes of early neural development, i.e. the specification and differentiation of neural crest cells. One explanation for these similarities would be that BMSC activate a transcriptional program that recapitulates early steps of neuronal development. Another possibility, which had to be proven in future, would be that BMSC are descendants of neural crest cells themselves. A neuroectodermal origin of BMSC could also explain their differentiation capacity and would raise a completely new perspective to investigate their developmental biology

Gene expression profile of mouse bone marrow stromal cells determined by cDNA microarray analysis

Bone marrow stromal cells (BMSC) have gained increased attention because of their multipotency and adult stem cell character. They have been shown to differentiate into other cell types of the mesenchymal lineage and also into non-mesenchymal cells. The exact identity of the original cells, which are isolated from bone marrow by their selective adherence to plastic, remains unknown to date. We have established and characterized mouse BMSC cultures and analyzed three independent samples by cDNA microarrays. The expression profile was compared with two previous expression studies of human BMSC and revealed a high degree of concordance between different techniques and species. To gain clues about the positional context and biology of the isolated cells within the bone marrow stroma, we searched our data for genes that encode proteins of the extracellular matrix, cell adhesion proteins, cytoskeletal proteins and cytokines/cytokine receptors. This analysis revealed a close association of BMSC with vascular cells and indicated that BMSC resemble pericytes

Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells.

BACKGROUND: Cross-sectional studies suggest that maternal exposure to farming decreases the risk of allergic diseases in offspring. The potential underlying immunologic mechanisms are not understood. OBJECTIVE: We sought to assess whether maternal farm exposure activates regulatory T (Treg) cells in cord blood, exerting T(H)2-suppressive effects after microbial stimulation. METHODS: Eighty-four pregnant mothers were recruited before delivery. Detailed questionnaires (60 nonfarming and 22 farming mothers with 2 exclusions) assessed the farming exposures. Cord blood was stimulated with the microbial stimulus peptidoglycan (Ppg), the mitogen PHA, house dust mite extracts (Der p 1), and combinations. Treg cells (CD4+CD25(high) cells; intracellular forkhead/winged-helix family transcriptional repressor p3 [FOXP3] expression, FOXP3 levels, lymphocyte activation gene 3 mRNA expression, functional studies, and DNA methylation of the FOXP3 locus), proliferation, and T(H)2/T(H)1/T(H)17 cytokines were examined. RESULTS: Cord blood Treg cell counts (both unstimulated and PHA stimulated) were increased with maternal farming exposures and associated with higher FOXP3 (Der p 1 + Ppg stimulation) and trendwise higher lymphocyte activation gene 3 (Ppg) expression. Furthermore, Treg cell function was more efficient with farming exposure (effector cell suppression, P = .004). In parallel, T(H)2 cytokine (IL-5) levels were decreased and associated with decreased lymphoproliferation and increased IL-6 levels (Ppg stimulation, Der p 1 + Ppg stimulation, or both; P < .05). Maternal exposure to increasing numbers of farm animals and stables was discovered to exert distinct effects on Treg cells, T(H)1/T(H)2 cells, or both. Additionally, FOXP3 demethylation in offspring of mothers with farm milk exposure was increased (P = .02). CONCLUSIONS: Farm exposures during pregnancy increase the number and function of cord blood Treg cells associated with lower T(H)2 cytokine secretion and lymphocyte proliferation on innate exposure. One fascinating speculation is that maternal farm exposure might reflect a natural model of immunotherapy, potentially including a selection of innate stimuli in addition to allergen, shaping a child's immune system at an early stage

The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory IL-1β and recently has become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1β pathway in granuloma formation and sarcoidosis.Nineteen sarcoid patients and 19 healthy volunteers (HV) were recruited into this pilot study. NLRP3 inflammasome activity was measured in BAL-cells and lung and skin biopsies using immunohistochemistry, Western blot, RT-PCR and ELISA. For in vivo experiments we used the trehalose 6,6 dimycolate (TDM)- granuloma mouse model and evaluated lung granuloma burden in miR-223 KO and NLRP3 KO mice as well as the treatment effects of MCC950 and anti-IL-1β antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1β in lung granuloma, and increased IL-1β release of BAL-cells from sarcoid patients compared to HV (p=0.006). mRNA levels of miR-223, a micro RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 KO mice showed decreased and miR-223 KO mice increased granuloma formation compared to wildtype. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1β antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1β pathway activation in sarcoidosis and suggest both as valid therapeutic targets

Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutanesous syndrome

Noonan syndrome (NS) is a relatively common, but genetically heterogeneous autosomal dominant malformation syndrome. Characteristic features are proportionate short stature, dysmorphic face, and congenital heart defects. Only recently, a gene involved in NS could be identified. It encodes the non-receptor protein tyrosine phosphatase SHP-2, which is an important molecule in several intracellular signal transduction pathways that control diverse developmental processes, most importantly cardiac semilunar valvulogenesis. We have screened this gene for mutations in 96 familial and sporadic, well-characterised NS patients and identified 15 different missense mutations in a total of 32 patients (33%), including 23 index patients. Most changes clustered in one exon which encodes parts of the N-SH2 domain. Five of the mutations were recurrent. Interestingly, no mutations in the PTPN11 gene were detected in five additional patients with cardio-facio-cutaneous (CFC) syndrome, which shows clinical similarities to NS

Expanding the phenotypic spectrum of lupus erythematosus in Aicardi-Goutières syndrome

Objective. Aicardi-Goutières syndrome (AGS) is an early-onset encephalopathy resembling congenital viral infection that is characterized by basal ganglia calcifications, loss of white matter, cerebrospinal fluid (CSF) lymphocytosis, and elevated interferon-␣ levels in the CSF. Studies have shown that AGS is an autosomalrecessive disease linked to mutations in 5 genes, encoding the 3-repair DNA exonuclease 1 (TREX1), the 3 subunits of ribonuclease H2 (RNASEH2A-C), and sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1). In this study we further characterized the phenotypic spectrum of this disease. Methods. Clinical and laboratory data were obtained from 26 patients fulfilling the clinical diagnostic criteria for AGS. Genomic DNA was screened for mutations in all 5 AGS genes by direct sequencing, and sera were analyzed for autoantibodies. Results. In 20 patients with AGS, 20 mutations, 12 of which were novel, were identified in all 5 AGS genes. Clinical and laboratory investigations revealed a high prevalence of features (some not previously described in patients with AGS) that are commonly seen in patients with systemic lupus erythematosus (SLE), such as thrombocytopenia, leukocytopenia, antinuclear antibodies, erythematous lesions, oral ulcers, and arthritis, which were observed in 12 (60%) of 20 patients with AGS. Moreover, the coexistence of AGS and SLE, was for the first time, demonstrated in 2 patients with molecularly proven AGS. Conclusion. These findings expand the phenotypic spectrum of lupus erythematosus in AGS and provide further insight into its disease mechanisms by Supported by the Deutsche Forschungsgemeinschaft (DFG grant LE 1074/3-1)