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Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and by their plasticity promote all phases of tumor development. Mechanisms of macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced immunosuppressive interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at identifying signaling pathways contributing to generation of TAM-like M Phi by using supernatant of apoptotic cancer cells (ACM) as stimulus.
To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral RNAi-based approach. The primary read-out was production of IL-10. However, mediators modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8 and IL-12. Following assay development, optimization and down-scaling to a 384-well format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion.
One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development, but compelling evidence emerges suggesting participation of TRKA in cancer development. First experiments using pharmacological inhibitors principally confirmed the involvement of TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser extend MAPK p38 as important signaling molecules downstream of TRKA activation. Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF neutralization experiments. NGF was not induced by or present in ACM, but was constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated by other receptors and in neurons its cellular localization is decisive for its function. Inhibitors of common transactivation partners did not influence IL-10 production by human M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I was intrigued to clarify mechanisms of TRKA trafficking in response to ACM.
The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore, inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA to the plasma membrane. Following the detailed analysis of IL-10 regulation, I was interested whether other TAM phenotype markers were influenced by ACM and whether their expression was regulated through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for induction of all but one marker, whereas TRKA signaling was only required for cytokine secretion. Interestingly, none of the investigated TAM markers was regulated identically to IL-10, emphasizing a tight and exclusive regulation machinery of this potent immunosuppressive cytokine.
Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I isolated murine TAM as well as other major mononuclear phagocyte populations from primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was required for spontaneous cytokine production selectively by primary murine TAM. Besides IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic protein-1, indicating its relevance in cancer-associated inflammation.
In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as NGF, might be interesting targets for future cancer therapy.
Objectives: To immunohistochemically characterize and correlate macrophage M1/M2 polarization status with disease severity at peri-implantitis sites.
Materials and methods: A total of twenty patients (n = 20 implants) diagnosed with peri-implantitis (i.e., bleeding on probing with or without suppuration, probing depths ≥ 6 mm, and radiographic marginal bone loss ≥ 3 mm) were included. The severity of peri-implantitis was classified according to established criteria (i.e., slight, moderate, and advanced). Granulation tissue biopsies were obtained during surgical therapy and prepared for immunohistological assessment and macrophage polarization characterization. Macrophages, M1, and M2 phenotypes were identified through immunohistochemical markers (i.e., CD68, CD80, and CD206) and quantified through histomorphometrical analyses.
Results: Macrophages exhibiting a positive CD68 expression occupied a mean proportion of 14.36% (95% CI 11.4–17.2) of the inflammatory connective tissue (ICT) area. Positive M1 (CD80) and M2 (CD206) macrophages occupied a mean value of 7.07% (95% CI 5.9–9.4) and 5.22% (95% CI 3.8–6.6) of the ICT, respectively. The mean M1/M2 ratio was 1.56 (95% CI 1–12–1.9). Advanced peri-implantitis cases expressed a significantly higher M1 (%) when compared with M2 (%) expression. There was a significant correlation between CD68 (%) and M1 (%) expression and probing depth (PD) values.
Conclusion: The present immunohistochemical analysis suggests that macrophages constitute a considerable proportion of the inflammatory cellular composition at peri-implantitis sites, revealing a significant higher expression for M1 inflammatory phenotype at advanced peri-implantitis sites, which could possibly play a critical role in disease progression.
Clinical relevance: Macrophages have critical functions to establish homeostasis and disease. Bacteria might induce oral dysbiosis unbalancing the host’s immunological response and triggering inflammation around dental implants. M1/M2 status could possibly reveal peri-implantitis’ underlying pathogenesis.
Background: Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells.
Aims: We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets.
Methods: Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used.
Results: Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented.
Conclusions: We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.
Inflammatory nontraumatic atlantoaxial rotatory subluxation (AAS) in children is an often-missed diagnosis, especially in the early stages of disease. Abscess formation and spinal cord compression are serious risks that call for immediate surgical attention. Neither radiographs nor non-enhanced computed tomography (CT) images sufficiently indicate inflammatory processes. Magnetic resonance imaging (MRI) allows a thorough evaluation of paraspinal soft tissues, joints, and ligaments. In addition, it can show evidence of vertebral distraction and spinal cord compression. After conducting a scoping review of the literature, along with scientific and practical considerations, we outlined a standardized pediatric MRI protocol for suspected inflammatory nontraumatic AAS. We recommend contrast-enhanced MRI as the primary diagnostic imaging modality in children with signs of torticollis in combination with nasopharyngeal inflammatory or ear nose and throat (ENT) surgical history.
Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal alpha-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.
Objective: Management and outcomes of superficial vein thrombosis (SVT) are highly variable and not well described. Therefore, the INvestigating SIGnificant Health TrendS in the management of SVT (INSIGHTS-SVT) study collected prospective data under real life conditions.
Methods: Prospective observational study of objectively confirmed acute isolated SVT. The primary outcome was a composite of symptomatic deep vein thrombosis (DVT), pulmonary embolism (PE), and extension or recurrence of SVT at three months. The primary safety outcome was clinically relevant bleeding.
Results: A total of 1 150 patients were included (mean age 60.2 ± 14.7 years; 64.9% women; mean BMI 29.4 ± 6.3 kg/m2). SVT was below the knee in 54.5%, above the knee in 26.7%, above and below the knee in 18.8%. At baseline, 93.6% received pharmacological treatment (65.7% fondaparinux, 23.2% heparins, 4.3% direct oral anticoagulants [DOACs], 14.5% analgesics), 77.0% compression treatment, and 1.9% surgery; 6.4% did not receive any anticoagulation. The primary outcome occurred in 5.8%; 4.7% had recurrent or extended SVT, 1.7% DVT, and 0.8% PE. Clinically relevant non-major bleeding occurred in 1.2% and major bleeding in 0.3%. Complete clinical recovery of SVT was reported in 708 patients (62.4%). Primary outcome adjusted by propensity score and for treatment duration was lower with fondaparinux compared with low molecular weight heparin (4.4% vs. 9.6%; hazard ratio [HR] 0.51; 95% confidence interval [CI] 0.3 - 0.9; p = .017). On multivariable analysis, associated factors for primary outcome included another SVT prior to the present SVT event (HR 2.3), age per year (HR 0.97), duration of drug treatment per week (HR 0.92), and thrombus length (HR 1.03).
Conclusion: At three month follow up, patients with isolated SVT are at risk of thromboembolic complications (mainly recurrent or extended SVT), despite anticoagulation. In this real life study, about one third had received either heparins, oral anticoagulants, or no anticoagulation.
Probably, patients with de novo (synchronous) and recurrent (metachronous) oligometastatic hormone-sensitive prostate cancer have different oncologic outcomes. Thus, we are challenged with different scenarios in clinical practice, where different treatment options may apply. In the last years, several prospective studies have focused on the treatment of patients with de novo oligometastatic hormone-sensitive prostate cancer. Not only the addition of systemic therapeutic treatments, such as chemotherapy with docetaxel, abiraterone, enzalutamide, and apalutamide, next to androgen deprivation therapy, demonstrated to improve outcomes in these patients but also local therapy of the primary has been demonstrated to improve outcomes of low-volume metastatic disease. Next to radiotherapy, also radical prostatectomy has been reported as a feasible and safe treatment option. Additional metastasis-directed therapy in de novo metastatic disease is currently examined by four trials. In the recurrent metastatic setting, less data are available, and it remains uncertain if patients can be treated in the same way as synchronous oligometastatic disease. Metastasis-directed therapy has demonstrated to prolong outcomes, while data on survival are still missing.
Background: Radiotherapy dose and target volume prescriptions for anal squamous cell carcinoma (ASCC) vary considerably in daily practice and guidelines, including those from NCCN, UK, Australasian, and ESMO. We conducted a pattern-of-care survey to assess the patient management in German speaking countries.
Methods: We developed an anonymous questionnaire comprising 18 questions on diagnosis and treatment of ASCC. The survey was sent to 361 DEGRO-associated institutions, including 41 university hospitals, 118 non-university institutions, and 202 private practices.
Results: We received a total of 101 (28%) surveys, including 20 (19.8%) from university, 36 (35.6%) from non-university clinics, and 45 (44.6%) from private practices. A total of 28 (27.8%) institutions reported to treat more than 5 patients with early-stage ASCC and 42 (41.6%) institutions treat more than 5 patients with locoregionally-advanced ASCC per year. Biopsy of suspicious inguinal nodes was advocated in only 12 (11.8%) centers. Screening for human immunodeficiency virus (HIV) is done in 28 (27.7%). Intensity modulated radiotherapy or similar techniques are used in 97%. The elective lymph node dose ranged from 30.6 Gy to 52.8 Gy, whereas 87% prescribed 50.4–55. 8 Gy (range: 30.6 to 59.4 Gy) to the involved lymph nodes. The dose to gross disease of cT1 or cT2 ASCC ranged from 50 to ≥60 Gy. For cT3 or cT4 tumors the target dose ranged from 54 Gy to more than 60 Gy, with 76 (75.2%) institutions prescribing 59.4 Gy. The preferred concurrent chemotherapy regimen was 5-FU/Mitomycin C, whereas 6 (6%) prescribed Capecitabine/Mitomycin C. HIV-positive patients are treated with full-dose CRT in 87 (86.1%) institutions. First assessment for clinical response is reported to be performed at 4–6 weeks after completion of CRT in 2 (2%) institutions, at 6–8 weeks in 20 (19.8%), and 79 (78%) institutions wait up to 5 months.
Conclusions: We observed marked differences in radiotherapy doses and treatment technique in patients with ASCC, and also variable approaches for patients with HIV. These data underline the need for an consensus treatment guideline for ASCC.
Objectives: To discuss optimal management of recurrent urinary tract infections (UTIs) in women. About every second woman experiences at least one UTI in her lifetime, of those 30% experience another UTI, and 3% further recurrences. Especially young healthy women without underlying anatomical deficiencies suffer from recurrent UTIs (rUTI), which are associated with significant morbidity and reduction in quality of life.
Methods: This is a narrative review, investigating publications dealing with recurrent UTI in women. Risk factors and options for management are discussed.
Results: The increased susceptibility of women to rUTI is based on the female anatomy in addition to behavioural, genetic, and urological factors. However, why some women are more likely than others to develop and maintain rUTI remains to be clarified. Invasive characteristics of certain uropathogenic Escherichia coli that are able to form extra- and intracellular biofilms and may therefore cause delayed release of bacteria into the bladder, may play a role in this setting. Treatment recommendations for an acute episode of rUTI do not differ from those for isolated episodes. Given the nature of rUTI, different prophylactic approaches also play an important role. Women with rUTI should first be counselled to use non-antibiotic strategies including behavioural changes, anti-adhesive treatments, antiseptics, and immunomodulation, before antibiotic prophylaxis is considered. In addition to the traditional treatment and prophylactic therapies, new experimental strategies are emerging and show promising effects, such as faecal microbiota transfer (FMT), a treatment option that transfers microorganisms and metabolites of a healthy donor’s faecal matter to patients using oral capsules, enemas, or endoscopy. Initial findings suggest that FMT might be a promising treatment approach to interrupt the cycle of rUTI. Furthermore, bacteriophages, infecting and replicating in bacteria, have been clinically trialled for UTIs.
Conclusion: Due to the limitation of available data, novel treatment options require further clinical research to objectify the potential in treating bacterial infections, particularly UTIs.
Purpose: Every physician must be able to sufficiently master medical emergencies, especially in medical areas where emergencies occur frequently such as in the emergency room or emergency surgery. This contrasts with the observation that medical students and young residents often feel insufficiently prepared to handle medical emergencies. It is therefore necessary to train them in the treatment of emergency patients. The aim of this study is to analyze the influence of the assignment of manikin versus simulated patients during a training for undergraduate medical students on learning outcomes and the perceived realism.
Methods: The study had a prospective cross-over design and took place in a 3-day emergency medicine training for undergraduate medical students. Students completed three teaching units (‘chest pain’, ‘impaired consciousness’, ‘dyspnea’), either with manikin or simulated patient. Using a questionnaire after each unit, overall impression, didactics, content, the quality of practical exercises, and the learning success were evaluated. The gained competences were measured in a 6-station objective structured clinical examination (OSCE) at the end of training.
Results: 126 students participated. Students rated simulated patients as significantly more realistic than manikins regarding the possibility to carry out examination techniques and taking medical history. 54.92% of the students would prefer to train with simulated patients in the future. Regarding the gained competences for ‘chest pain’ and ‘impaired consciousness’, students who trained with a manikin scored less in the OSCE station than the simulated patients-group.
Conclusion: Simulated patients are rated more realistic than manikins and seem to be superior to manikins regarding gained competence.
The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.
Encouraging clinical results were reported on a novel cone-in-cone coupling for the fixation of dental implant-supported crowns (Acuris, Dentsply Sirona Implants, Mölndal, Sweden). However, the presence or absence of a microgap and a potential bacterial leakage at the conometric joint has not yet been investigated. A misfit and a resulting gap between the conometric components could potentially serve as a bacterial reservoir that promotes plaque formation, which in turn may lead to inflammation of the peri-implant tissues. Thus, a two-fold study set-up was designed in order to evaluate the bidirectional translocation of bacteria along conometrically seated single crowns. On conometric abutments filled with a culture suspension of anaerobic bacteria, the corresponding titanium nitride-coated (TiN) caps were fixed by friction. Each system was sterilized and immersed in culture medium to provide an optimal environment for microbial growth. Positive and negative controls were prepared. Specimens were stored in an anaerobic workstation, and total and viable bacterial counts were determined. Every 48 h, samples were taken from the reaction tubes to inoculate blood agar plates and to isolate bacterial DNA for quantification using qrt-PCR. In addition, one Acuris test system was subjected to scanning electron microscopy (SEM) to evaluate the precision of fit of the conometric coupling and marginal crown opening. Throughout the observational period of one week, blood agar plates of the specimens showed no viable bacterial growth. qrt-PCR, likewise, yielded a result approaching zero with an amount of about 0.53 × 10−4 µg/mL DNA. While the luting gap/marginal opening between the TiN-cap and the ceramic crown was within the clinically acceptable range, the SEM analysis failed to identify a measurable microgap at the cone-in-cone junction. Within the limits of the in-vitro study it can be concluded that the Acuris conometric interface does not allow for bacterial translocation under non-dynamic loading conditions.
Marjan van den Akker, Gesundheitswissenschaftlerin und Epidemiologin : Goethe, Deine Forscher
(2024)
Quantitative MRI allows to probe tissue properties by measuring relaxation times and may thus detect subtle changes in tissue composition. In this work we analyzed different relaxation times (T1, T2, T2* and T2′) and histological features in 321 samples that were acquired from 25 patients with newly diagnosed IDH wild-type glioma. Quantitative relaxation times before intravenous application of gadolinium-based contrast agent (GBCA), T1 relaxation time after GBCA as well as the relative difference between T1 relaxation times pre-to-post GBCA (T1rel) were compared with histopathologic features such as the presence of tumor cells, cell and vessel density, endogenous markers for hypoxia and cell proliferation. Image-guided stereotactic biopsy allowed for the attribution of each tissue specimen to its corresponding position in the respective relaxation time map. Compared to normal tissue, T1 and T2 relaxation times and T1rel were prolonged in samples containing tumor cells. The presence of vascular proliferates was associated with higher T1rel values. Immunopositivity for lactate dehydrogenase A (LDHA) involved slightly longer T1 relaxation times. However, low T2′ values, suggesting high amounts of deoxyhemoglobin, were found in samples with elevated vessel densities, but not in samples with increased immunopositivity for LDHA. Taken together, some of our observations were consistent with previous findings but the correlation of quantitative MRI and histologic parameters did not confirm all our pathophysiology-based assumptions.
Women with thrombophilic defects have been shown to be at increased risk, not only of pregnancy associated thromboembolism but also of other vascular complications of pregnancy, including preeclampsia and fetal loss. First trimester fetal loss is associated with factor V Leiden mutation, activated protein C resistance without factor V Leiden mutation and prothrombin G20210A mutation. Late nonrecurrent fetal loss is associated with factor V Leiden mutation, prothrombin mutation and protein S deficiency. Concerning acquired thrombophilia, recurrent fetal loss is a well-documented finding in patients with antiphospholipid antibodies. Associations between thrombophilia polymorphisms and an increased risk of intrauterine growth restriction have been discussed in small series of cases but could not be confirmed in large scale studies. Frequencies for anticardiolipin antibodies or lupus anticoagulants and antinuclear antibodies were significantly higher in women with infants small for gestational age compared to controls. Concerning preeclampsia, gestational hypertension and thrombophilia, a number of studies have examined these relationships with conflicting results. For factor V Leiden, MTHFR C677T and prothrombin mutation, no association with preeclampsia was observed, when severe cases were excluded. If studies were restricted to those of severe preeclampsia, an association with the factor V Leiden mutation was apparent and, to a lesser extent, with the MTHFR-mutation. For antithrombotic therapy, it was shown that in women with antiphospholipid syndrome and recurrent pregnancy loss, unfractionated heparin plus lowdose aspirin results in significantly better gestational outcome than lowdose aspirin alone. Concerning therapy of women with inherited thrombophilia and pregnancy loss, only small, uncontrolled studies are available, demonstrating improved pregnancy outcome when low molecular weight heparin (LMWH) is used for treatment. In conclusion, heritable thrombophilia and the antiphospholipid-syndrome are major causes of fetal loss after exclusion of other underlying pathologies like chromosomal abnormalities, and screening should be recommended. LMWH with or without aspirin may be used for treatment. There is little value in antenatal screening for prothrombotic polymorphisms to predict the development of small for gestational age infants, preeclampsia or gestational hypertension.
Mathematical modeling of the molecular switch of TNFR1-mediated signaling pathways using Petri nets
(2021)
The paper describes a mathematical model of the molecular switch of cell survival, apoptosis, and necroptosis in cellular signaling pathways initiated by tumor necrosis factor 1. Based on experimental findings in the current literature, we constructed a Petri net model in terms of detailed molecular reactions for the molecular players, protein complexes, post-translational modifications, and cross talk. The model comprises 118 biochemical entities, 130 reactions, and 299 connecting edges. Applying Petri net analysis techniques, we found 279 pathways describing complete signal flows from receptor activation to cellular response, representing the combinatorial diversity of functional pathways.120 pathways steered the cell to survival, whereas 58 and 35 pathways led to apoptosis and necroptosis, respectively. For 65 pathways, the triggered response was not deterministic, leading to multiple possible outcomes. Based on the Petri net, we investigated the detailed in silico knockout behavior and identified important checkpoints of the TNFR1 signaling pathway in terms of ubiquitination within complex I and the gene expression dependent on NF-κB, which controls the caspase activity in complex II and apoptosis induction.
Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2- hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro, which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations.
Background: Treatment of acute stroke is highly time-dependent and performed by a multiprofessional, interdisciplinary team. Interface problems are expectable and issues relevant to patient safety are omnipresent. The Safety Attitudes Questionnaire (SAQ) is a validated and widely used instrument to measure patient safety climate. The objective of this study was to evaluate the SAQ for the first time in the context of acute stroke care. Methods: A survey was carried out during the STREAM trial (NCT 032282) at seven university hospitals in Germany from October 2017 to October 2018. The anonymous survey included 33 questions (5-point Likert scale, 1 = disagree to 5 = agree) and addressed the entire multiprofessional stroke team. Statistical analyses were used to examine psychometric properties as well as descriptive findings. Results: 164 questionnaires were completed yielding a response rate of 66.4%. 67.7% of respondents were physicians and 25.0% were nurses. Confirmatory Factor Analysis revealed that the original 6-factor structure fits the data adequately. The SAQ for acute stroke care showed strong internal consistency (α = 0.88). Exploratory analysis revealed differences in scores on the SAQ dimensions when comparing physicians to nurses and when comparing physicians according to their duration of professional experience. Conclusion: The SAQ is a helpful and well-applicable tool to measure patient safety in acute stroke care. In comparison to other high-risk fields in medicine, patient safety climate in acute stroke care seems to be on a similar level with the potential for further improvements. Trial registration: www.ClinicalTrials.gov Identifier: NCT032282.
Introduction: Improvements in both musculoskeletal and non-musculoskeletal manifestations are important treatment goals in psoriatic arthritis (PsA). Objective: These post hoc analyses determined whether additional benefits related to various PsA domains are observed in patients simultaneously achieving 50% improvement in American College of Rheumatology criteria (ACR50) and 100% improvement in Psoriasis Area Severity Index (PASI100), the primary endpoint of the SPIRIT-H2H study. Methods: Patients with active PsA and psoriasis in SPIRIT-H2H (N = 566) were categorised into two sets of four response groups irrespective of treatment allocation (approved dosages of ixekizumab or adalimumab): patients who simultaneously achieved ACR50 and PASI100 response, achieved ACR50 response only, achieved PASI100 response only, or did not achieve ACR50 or PASI100 response after 24 and 52 weeks of treatment. Patients achieving simultaneous ACR50 and PASI100 response were compared with the other patient response groups at the corresponding time point for efficacy and health-related quality of life (HRQoL) outcomes. Results: Patients simultaneously achieving ACR50 and PASI100 responses at week 24 or 52 showed higher rates of ACR70 response, minimal disease activity, Disease Activity in Psoriatic Arthritis ≤ 4, resolution of enthesitis and dactylitis, and HRQoL improvement at weeks 24 and 52, respectively, than the other corresponding response groups at both time points. Conclusion: High levels of disease control, such as those obtained with simultaneous achievement of ACR50 and PASI100 response, were linked to better outcomes across a wide range of endpoints that are important for patients with PsA. Patients meeting this combined endpoint showed more comprehensive and thus greater control of disease activity.
Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis.
Background: Mechanical thrombectomy and systemic thrombolysis are important therapies for stroke patients. However, there is disagreement about the accompanying risk of acute symptomatic seizures.
Methods: A retrospective analysis of patients with an acute ischaemic stroke caused by large vessel occlusion was performed. The patients were divided into four groups based on whether they received either mechanical thrombectomy (MT) or systemic thrombolysis (ST; group 1: MT+/ST−; group 2: MT+/ST+; group 3: MT−/ST+; group 4: MT−/ST−). Propensity score matching was conducted for each group combination (1:3, 1:4, 2:3, 2:4, 1:2, 3:4) using the covariates “NIHSS at admission”, “mRS prior to event” and “age”. The primary endpoint was defined as the occurrence of acute symptomatic seizures.
Results: A total of 987 patients met the inclusion criteria, of whom 208, 264, 169 and 346 belonged to groups 1, 2, 3 and 4, respectively. Propensity score matched groups consisted of 160:160, 143:143, 156:156, 144:144, 204:204 and 165:165 patients for the comparisons 1:3, 1:4, 2:3, 2:4, 1:2 and 3:4, respectively. Based on chi-squared tests, there was no significant difference in the frequency of acute symptomatic seizures between the groups. Subgroups varied in their frequency of acute symptomatic seizures, ranging from 2.8 to 3.8%, 2.8–4.4%, 3.6–3.8% and 4.9–6.3% in groups 1, 2, 3 and 4, respectively.
Conclusion: There was no association between MT or ST and an increased risk of acute symptomatic seizures in patients with an acute ischaemic stroke caused by large vessel occlusion who were treated at a primary stroke centre.
Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease of the hair follicles leading to painful lesions, associated with increased levels of pro-inflammatory cytokines. Numerous guidelines recommend antibiotics like clindamycin and rifampicin in combination, as first-line systemic therapy in moderate-to-severe forms of inflammation. HS has been proposed to be mainly an auto-inflammatory disease associated with but not initially provoked by bacteria. Therefore, it has to be assumed that the pro-inflammatory milieu previously observed in HS skin is not solely dampened by the bacteriostatic inhibition of DNA-dependent RNA polymerase. To further clarify the mechanism of anti-inflammatory effects of rifampicin, ex vivo explants of lesional HS from 8 HS patients were treated with rifampicin, and its effect on cytokine production, immune cells as well as the expression of Toll-like receptor 2 (TLR2) were investigated. Analysis of cell culture medium of rifampicin-treated HS explants revealed an anti-inflammatory effect of rifampicin that significantly inhibiting interleukin (IL)-1β, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)-α production. Immunohistochemistry of the rifampicin-treated explants suggested a tendency for it to reduce the expression of TLR2 while not affecting the number of immune cells.
The endoplasmic reticulum (ER) is the largest cellular organelle that undergoes constant turnover upon diverse functional demands and cellular signals. Removal of nonfunctional or superfluous subdomains is balanced by the parallel expansion and formation of ER membranes, leading to the dynamic exchange of ER components. In recent years, selective autophagy of the ER, termed ER-phagy, has emerged as a predominant process involved in ER degradation and maintenance of ER homeostasis. Identification of multiple ER-phagy receptors, many with additional ER-shaping functions, paved the way for our molecular understanding of ER turnover in different cells and organs. In this review, we describe the molecular principles underling the physiological functions of ER-phagy in maintaining ER homeostasis via receptor-mediated macroautophagy and elaborate current focus points of the field.
IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
The ICH M13A draft bioequivalence guideline allows the exclusion of very low plasma profiles from the statistical evaluation in exceptional cases, i.e., if such phenomenon occurs due to non-compliance of subjects (not swallowing the product). Moreover, the draft ICH guideline requests additional bioequivalence studies for medicinal products with pH-dependent solubility after concomitant administration of gastric pH modifying preparations, e.g., proton pump inhibitors. Both regulations are scientifically sound, however, would need further specification. Main problem in this context is that compounds with very low solubility and slow intrinsic dissolution in the intestinal environment will cause significant bioavailability problems if their solid oral dosage forms are emptied from the stomach undisintegrated. Also very low plasma profiles may result under these circumstances. Such cases can occur accidentally and are not resultant of non-compliance. Thus, limitation for one case per study only as suggested in the guideline is not justified.
Meningioma surgery in patients ≥70 years of age: clinical outcome and validation of the SKALE score
(2021)
Along with increasing average life expectancy, the number of elderly meningioma patients has grown proportionally. Our aim was to evaluate whether these specific patients benefit from surgery and to investigate a previously published score for decision-making in meningioma patients (SKALE). Of 421 patients who underwent primary intracranial meningioma resection between 2009 and 2015, 71 patients were ≥70 years of age. We compared clinical data including World Health Organization (WHO) grade, MIB-1 proliferation index, Karnofsky Performance Status Scale (KPS), progression free survival (PFS) and mortality rate between elderly and all other meningioma patients. Preoperative SKALE scores (Sex, KPS, ASA score, location and edema) were determined for elderly patients. SKALE ≥8 was set for dichotomization to determine any association with outcome parameters. In 71 elderly patients (male/female 37/34) all data were available. Postoperative KPS was significantly lower in elderly patients (p < 0.0001). Pulmonary complications including pneumonia (10% vs. 3.2%; p = 0.0202) and pulmonary embolism (12.7% vs. 6%; p = 0.0209) occurred more frequently in our elderly cohort. Analyses of the Kaplan Meier curves revealed differences in three-month (5.6% vs. 0.3%; p = 0.0033), six-month (7% vs. 0.3%; p = 0.0006) and one-year mortality (8.5% vs. 0.3%; p < 0.0001) for elderly patients. Statistical analysis showed significant survival benefit in terms of one-year mortality for elderly patients with SKALE scores ≥8 (5.1 vs. 25%; p = 0.0479). According to our data, elderly meningioma patients face higher postoperative morbidity and mortality than younger patients. However, resection is reasonable for selected patients, particularly when reaching a SKALE score ≥ 8.
Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.
There has been a renewed interest in the potential use of psychedelics for the treatment of psychiatric conditions. Nevertheless, little is known about the mechanism of action and molecular pathways influenced by ayahuasca use in humans. Therefore, for the first time, our study aims to investigate the human metabolomics signature after consumption of a psychedelic, ayahuasca, and its connection with both the psychedelic-induced subjective effects and the plasma concentrations of ayahuasca alkaloids.
Plasma samples of 23 individuals were collected both before and after ayahuasca consumption. Samples were analysed through targeted metabolomics and further integrated with subjective ratings of the ayahuasca experience (i.e., using the 5-Dimension Altered States of Consciousness Rating Scale [ASC]), and plasma ayahuasca-alkaloids using integrated network analysis. Metabolic pathways enrichment analysis using diffusion algorithms for specific KEGG modules was performed on the metabolic output.
Compared to baseline, the consumption of ayahuasca increased N-acyl-ethanolamine endocannabinoids, decreased 2-acyl-glycerol endocannabinoids, and altered several large-neutral amino acids (LNAAs). Integrated network results indicated that most of the LNAAs were inversely associated with 9 out of the 11 subscales of the ASC, except for tryptophan which was positively associated. Several endocannabinoids and hexosylceramides were directly associated with the ayahuasca alkaloids. Enrichment analysis confirmed dysregulation in several pathways involved in neurotransmission such as serotonin and dopamine synthesis.
In conclusion, a crosstalk between the circulating LNAAs and the subjective effects is suggested, which is independent of the alkaloid concentrations and provides insights into the specific metabolic fingerprint and mechanism of action underlying ayahuasca experiences.
Hintergrund und Fragestellung: Patientensicherheit ist in den letzten Jahren zum intensiv diskutierten Thema geworden. Zudem rückt als potenzielle Basis der Patientensicherheit die Patientensicherheitskultur von Einrichtungen des Gesundheitswesens in den Fokus, bislang wurde dahingehend vor allem der stationäre Bereich untersucht. Ziel dieser Arbeit ist es, einen Einblick in den aktuellen Stand der Patientensicherheitskultur in Hausarztpraxen zu geben, und diesbezügliche Einflussfaktoren und Zusammenhänge aufzuzeigen. Dabei wurden insbesondere zwei Fragestellungen untersucht: 1. Lässt sich ein Zusammenhang aufzeigen zwischen einzelnen Praxis- und Teammerkmalen einerseits und den Ergebnissen in den Bereichen Sicherheitsklima, Patientensicherheitsindikatoren und Fehlermanagement andererseits? 2. Lassen sich durch einzelne Praxis- und Teammerkmale bzw. Sicherheitsklimafaktoren die Ausprägungen einer Praxis in bestimmten Patientensicherheitsindikatoren und dem Fehlermanagement einer Praxis vorhersagen?
Material und Methoden: In 60 allgemeinärztlich tätigen Praxen aus Hessen wurde die Patientensicherheitskultur anhand von drei Methoden gemessen. Dies waren 1. der auf Selbstauskunft beruhende „Fragebogen zum Sicherheitsklima“, 2. durch Praxisbegehungen und Interviews erfasste Patientensicherheitsindikatoren, sowie 3. detailliert analysierte Fehlerberichte. Die statistische Auswertung umfasste u.a. Korrelationsanalysen (Mann-Whitney-U, Wilcoxon-W, Spearman-Rangkorrelation) sowie multivariate schrittweise Regressionsanalysen.
Ergebnisse: Die Beurteilung des Sicherheitsklimas fiel über alle Praxen hinweg homogen positiv aus (acht von neun Sicherheitsklimafaktoren mit Mittelwerten von mind. vier von fünf Punkten). Bei den 12 Patientensicherheitsindikatoren ergaben sich differenziertere Werte (niedrigster Mittelwert: Indikator „Marcumartherapie“ mit 0,43 von 1, höchster Mittelwert: Indikator „Allergiehinweis“ mit 0,75 von 1). Es gingen 24 Berichte kritischer Ereignisse ein, die zu 79% als „Kein Fehlermanagement“ oder „Unbefriedigendes Fehlermanagement“ beurteilt wurden. Die Korrelations- und Regressionsanalysen zeigten Zusammenhänge auf, z.B. erzielten größere Praxisteams niedrigere Werte beim Patientensicherheitsklima und höhere Werte bei den Patientensicherheitsindikatoren im Vergleich zum Durchschnitt.
Diskussion: Sicherheitsklima, Patientensicherheitsindikatoren und Fehlermanagement sind in einer Hausarztpraxis mit den verwendeten Instrumenten messbar. Jedes der drei Instrumente misst einen anderen, wichtigen Bereich der Sicherheitskultur, wodurch jeweils unterschiedliche Einstellungen und Prozesse beleuchtet und anschließend auch beurteilt und verbessert werden können. In den Analysen zur Beantwortung der beiden Fragestellungen konnten Zusammenhänge und Vorhersagevariablen herausgearbeitet werden, allerdings waren diese Zusammenhänge zum Teil entgegengesetzt. Daraus ergibt sich die Hypothese, dass Praxis- und Teammerkmale als Voraussetzungen zu unterschiedlichen Ausprägungen von Sicherheitsklima, Patientensicherheitsindikatoren und Fehlermanagement führen können. Insgesamt könnte die Qualität der hausärztlichen Arbeit und die Sicherheit der Versorgung durch eine regelmäßige Reflektion der Praxisabläufe anhand der drei Messmethoden gesteigert werden.
Die Diagnostik der pathologischen Proteinurie ist im Wandel begriffen. Während zur Zeit noch der Streifentest und die Gesamteiweißbestimmung in der Proteindiagnostik von Nierenerkrankungen im Vordergrund stehen, gewinnt die quantitative Bestimmung von Plasmaproteinen im Harn eine gewisse Bedeutung. Ursachen sind, neben einer mangelnden diagnostischen und analytischen Sensitiv/tat und Spezifität des Streifentests und der Gesamteiweißbestimmungsmethoden, der technische Fortschritt in der mechanisierten immunnephelometrischen oder immunturbidimetrischen Analytik der Plasmaproteine im Harn.
The MICOS complex subunit MIC13 is essential for mitochondrial cristae organization. Mutations in MIC13 cause severe mitochondrial hepato-encephalopathy displaying defective cristae morphology and loss of the MIC10-subcomplex. Here we identified stomatin-like protein 2 (SLP2) as an interacting partner of MIC13 and decipher a critical role of SLP2 as an auxiliary MICOS subunit, modulating cristae morphology. SLP2 provides a large interaction hub for MICOS subunits and loss of SLP2 leads to drastic alterations in cristae morphology. Double deletion of SLP2 and MIC13 showed reduced assembly of core MICOS subunit, MIC60 into MICOS and dispersion of MIC60-specific puncta, demonstrating a critical role of SLP2-MIC13 in MICOS assembly and crista junction (CJ) formation. We further identified that the mitochondrial i-AAA protease YME1L in coordination either with MIC13 or SLP2 differentially regulates MICOS assembly pathways thereby interlinking MIC13-specific or scaffolding-specific role of SLP2 with quality control and assembly of the MICOS complex. YME1L- depletion in MIC13 KO could restore MIC10-subcomplex and reform the nascent CJ. Taken together, we propose ‘seeder’ model for MICOS assembly and CJ formation, where SLP2- MIC13 seed the assembly of MIC60 into MICOS complex and promote the formation of CJ by regulating the quality and stability of MIC10-subcomplex.
MicroRNA miR-181 - a rheostat for TCR signaling in thymic selection and peripheral T-Cell function
(2020)
The selection of T cells during intra-thymic d evelopment is crucial to obtain a functional and simultaneously not self-reactive peripheral T cell repertoire. However, selection is a complex process dependent on T cell receptor (TCR) thresholds that remain incompletely understood. In peripheral T cells, activation, clonal expansion, and contraction of the active T cell pool, as well as other processes depend on TCR signal strength. Members of the microRNA (miRNA) miR-181 family have been shown to be dynamically regulated during T cell development as well as dependent on the activation stage of T cells. Indeed, it has been shown that expression of miR-181a leads to the downregulation of multiple phosphatases, implicating miR-181a as ‘‘rheostat’’ of TCR signaling. Consistently, genetic models have revealed an essential role of miR-181a/b-1 for the generation of unconventional T cells as well as a function in tuning TCR sensitivity in peripheral T cells during aging. Here, we review these broad roles of miR-181 family members in T cell function via modulating TCR signal strength.
Patients with type 2 diabetes (T2D) are threatened by excessive cardiovascular morbidity and mortality. While accelerated arterial stiffening may represent a critical mechanistic factor driving cardiovascular risk in T2D, specific therapies to contain the underlying diabetic arterial remodeling have been elusive. The present translational study investigates the role of microRNA-29b (miR-29b) as a driver and therapeutic target of diabetic aortic remodeling and stiffening. Using a murine model (db/db mice), as well as human aortic tissue samples, we find that diabetic aortic remodeling and stiffening is associated with medial fibrosis, as well as fragmentation of aortic elastic layers. miR-29b is significantly downregulated in T2D and miR-29b repression is sufficient to induce both aortic medial fibrosis and elastin breakdown through upregulation of its direct target genes COL1A1 and MMP2 thereby increasing aortic stiffness. Moreover, antioxidant treatment restores aortic miR-29b levels and counteracts diabetic aortic remodeling. Concluding, we identify miR-29b as a comprehensive—and therefore powerful—regulator of aortic remodeling and stiffening in T2D that moreover qualifies as a (redox-sensitive) target for therapeutic intervention.
Several microRNAs (miRNAs) are associated with the molecular pathogenesis of hepatocellular carcinoma (HCC). However, previous studies analyzing the dysregulation of miRNAs in HCC show heterogeneous results. We hypothesized that part of this heterogeneity might be attributable to variations of miRNA expression deriving from the HCC capsule or the fibrotic septa within the peritumoral tissue used as controls. Tissue from surgically resected hepatitis C–associated HCC from six well-matched patients was microdissected using laser microdissection and pressure catapulting technique. Four distinct histologic compartments were isolated: tumor parenchyma (TP), fibrous capsule of the tumor (TC), tumor-adjacent liver parenchyma (LP), and cirrhotic septa of the tumor-adjacent liver (LC). MiRNA expression profiling analysis of 1105 mature miRNAs and precursors was performed using miRNA microarray. Principal component analysis and consecutive pairwise supervised comparisons demonstrated distinct patterns of expressed miRNAs not only for TP versus LP (e.g., intratumoral down-regulation of miR-214, miR-199a, miR-146a, and miR-125a; P< .05) but also for TC versus LC (including down-regulation within TC of miR-126, miR-99a/100, miR-26a, and miR-125b; P< .05). The tumor capsule therefore demonstrates a tumor-like phenotype with down-regulation of well-known tumor-suppressive miRNAs. Variations of co-analyzed fibrotic tissue within the tumor or in controls may have profound influence on miRNA expression analyses in HCC. Several miRNAs, which are proposed to be HCC specific, may indeed be rather associated to the tumor capsule. As miRNAs evolve to be important biomarkers in liver tumors, the presented data have important translational implications on diagnostics and treatment in patients with HCC.
Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN.
Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.
Highlights
• Microstimulation of visual area V4 improves visual stimulus detection
• Effects of V4 microstimulation extend to the other hemifield
• Microstimulation effects are time dependent and consistent with attention dynamics
Summary
Neuronal activity in visual area V4 is well known to be modulated by selective attention, and there are reports on V4 lesions leading to attentional deficits. However, it remains unclear whether V4 microstimulation can elicit attentional benefits. To test this hypothesis, we performed local microstimulation in area V4 and explored its spatial and time dynamics in two macaque monkeys performing a visual detection task. Microstimulation was delivered via chronically implanted multi-electrode arrays. We found that microstimulation increases average performance by 35% and reduces luminance detection thresholds by −30%. This benefit critically depends on the onset of microstimulation relative to the stimulus, consistent with known dynamics of endogenous attention. These results show that local microstimulation of V4 can improve behavior and highlight the critical role of V4 for attention.
Purpose: To investigate cortical thickness and cortical quantitative T2 values as imaging markers of microstructural tissue damage in patients with unilateral high-grade internal carotid artery occlusive disease (ICAOD).
Methods: A total of 22 patients with ≥70% stenosis (mean age 64.8 years) and 20 older healthy control subjects (mean age 70.8 years) underwent structural magnetic resonance imaging (MRI) and high-resolution quantitative (q)T2 mapping. Generalized linear mixed models (GLMM) controlling for age and white matter lesion volume were employed to investigate the effect of ICAOD on imaging parameters of cortical microstructural integrity in multivariate analyses.
Results: There was a significant main effect (p < 0.05) of the group (patients/controls) on both cortical thickness and cortical qT2 values with cortical thinning and increased cortical qT2 in patients compared to controls, irrespective of the hemisphere. The presence of upstream carotid stenosis had a significant main effect on cortical qT2 values (p = 0.01) leading to increased qT2 in the poststenotic hemisphere, which was not found for cortical thickness. The GLMM showed that in general cortical thickness was decreased and cortical qT2 values were increased with increasing age (p < 0.05).
Conclusion: Unilateral high-grade carotid occlusive disease is associated with widespread cortical thinning and prolongation of cortical qT2, presumably reflecting hypoperfusion-related microstructural cortical damage similar to accelerated aging of the cerebral cortex. Cortical thinning and increase of cortical qT2 seem to reflect different aspects and different pathophysiological states of cortical degeneration. Quantitative T2 mapping might be a sensitive imaging biomarker for early cortical microstructural damage.
Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.
Das kolorektale Karzinom stellt die zweithäufigste Krebstodesursache bei Männern und Frauen in der Bundesrepublik Deutschland dar
Das CRC hat aus diesem Grund eine große Bedeutung in chirurgischen und radiologischen Fachgebieten. Hierbei spielen zahlreiche Verfahren und Behandlungsmethoden eine zentrale Rolle, um das CRC und die hiervon ausgehenden kolorektalen Lebermetastasen zu behandeln und eine bestmögliche Therapie zu evaluieren. Über die letzten Jahrzehnte haben sich daher viele verschiedene Methoden für die Behandlung von CRLMs entwickelt, wie Mikrowellenablation (MWA), laserinduzierte interstitielle Thermotherapie (LITT), Radiofrequenzablation (RFA) und das chirurgische Vorgehen. Die vielversprechendste unter den Techniken und Verfahren stellt die chirurgische Resektion dar. Problematisch ist hierbei, dass viele erkrankte Patienten keine ausreichend gute körperliche Verfassung mehr aufweisen, um eine Resektion ohne große Risiken durchführen zu können.
Das Hauptziel dieser Studie war es nun, eine möglichst genaue und
aussagekräftige Untersuchung von Patientengruppen durchzuführen, bei denen eine kolorektale Lebermetastase diagnostiziert wurde. In der vorliegenden Studie wurden 132 Patienten mit kolorektalen Lebermetastasen (CRLM) untersucht, welche zwischen 2010 und 2018 mit einer CT-gesteuerten MWA-Therapie im Institut für Diagnostische und Interventionelle Radiologie des Universitätsklinikums in Frankfurt am Main behandelt wurden. Hierbei war von besonderer Bedeutung, welche prognostischen Parameter die Überlebenszeiten und Überlebensraten beeinflussen. Die Daten konnten anhand von vielfältigen Personendaten und den dazugehörigen Therapieverläufen erhoben werden. Außerdem wurden CT-Bilder, welche im Zuge der Behandlung entstanden waren, für die Erhebung zusätzlicher Parameter verwendet. Die erhobenen Daten und Messwerte wurden retrospektiv ermittelt und umfassten eine große Patientengruppe. Dies steigert die Aussagekraft der Ergebnisse und Kennzahlen wesentlich. Ein besonderes Augenmerk lag auf der Einteilung der Patienten in zwei Gruppen entsprechend ihrer Behandlungsindikation.
Zu den prognostischen Faktoren zählten das Ablationssystem, die Lokation der Metastasen, die Anzahl der Metastasen, der technische Erfolg, die Energie und Leistung, der Durchmesser und das Volumen der Metastasen, die Vor- und Nachbehandlung und die Lokalrezidive.
Die Patientengruppe mit palliativer Therapieindikation (1.08 Jahre) zeigte eine signifikant geringere mediane Überlebenszeit im Vergleich mit der kurativen Patientengruppe (3.48 Jahre). Die mediane Überlebenszeit aller Patienten betrug insgesamt 2.68 Jahre. Zusätzlich wurden die Überlebensraten der Patienten ermittelt. Die 1- und 3-Jahres-Überlebensraten aller behandelten Patienten im Untersuchungszeitraum lagen bei 82.7% und 41.6%. Die 1- und 3-JahresÜberlebensraten der 57 Patienten mit palliativer Behandlungsindikation waren 54.4% und 14.9%. Im Vergleich hierzu betrugen die 1- und 3-JahresÜberlebensraten der kurativ behandelten Patientengruppe 96.9% und 55.1%. Die mediane Beobachtungszeit nach der Behandlung betrug 2.39 Jahre. In dieser Zeit erreichten 96.2% aller Patienten eine lokale Tumorkontrolle (127/132). Die Überlebenszeit von Patienten mit einer, zwei oder drei, vier oder fünf und multiplen Lebermetastasen betrug 3.79, 2.13, 1.09 und 0.93 Jahre (alle p<0,017). Es gab eine einzige relevante Komplikation (Abszess) bei allen Behandlungen (1/257; 0,4%). Alle Unterschiede der Überlebenszeiten im primären Tumorursprung (p <0,038) und bei der Anzahl der Metastasen waren signifikant. Die anderen prognostischen Faktoren zeigten keine statistische Signifikanz. Prognostische Faktoren wie die Anzahl der Lebermetastasen, die Lokation des Primärtumors und das verwendete Ablationssystem haben einen bedeutenden Einfluss auf die Überlebenszeiten der CRLM-Patienten in dieser Studie gezeigt. Die Ergebnisse dieser Studie sind als vornehmlich anzusehen, weil eine strenge Zuteilung der Patienten in kurative und palliative Behandlungsindikationen für die Analyse der Überlebensdaten in dieser Form bis zu diesem Zeitpunkt nicht durchgeführt worden war.
Die Prognosefaktoren und deren Einfluss auf die Überlebenszeiten stellen für zukünftige radiologische Prognosen und Therapiemaßnahmen in Bezug auf CRLM Patienten gute Richtwerte dar. Sowohl für die Radiologen und Ärzte als auch für die Patienten und Angehörigen sind dies zukunftsweisende Anhaltspunkte.
Mild acquired factor XIII deficiency and clinical relevance at the ICU - a retrospective analysis
(2021)
Acquired FXIII deficiency is a relevant complication in the perioperative setting; however, we still have little evidence about the incidence and management of this rarely isolated coagulopathy. This study aims to help find the right value for the substitution of patients with an acquired mild FXIII deficiency. In this retrospective single-center cohort study, we enrolled critically ill patients with mild acquired FXIII deficiency (>5% and ≤70%) and compared clinical and laboratory parameters, as well as pro-coagulatory treatments. The results of the present analysis of 104 patients support the clinical relevance of FXIII activity out of the normal range. Patients with lower FXIII levels, beginning at <60%, had lower minimum and maximum hemoglobin values, corresponding to the finding that patients with a minimum FXIII activity of <50% needed significantly more packed red blood cells. FXIII activity correlated significantly with general coagulation markers such as prothrombin time, activated partial thromboplastin time, and fibrinogen. Nevertheless, comparing the groups with a cut-off of 50%, the amount of fresh frozen plasma, thrombocytes, PPSB, AT-III, and fibrinogen given did not differ. These results indicate that a mild FXIII deficiency occurring at any point of intensive care unit stay is also probably relevant for the total need of packed red blood cells, independent of pro-coagulatory management. In alignment with the ESAIC guidelines, the measurement of FXIII in critically ill patients with the risk of bleeding and early management, with the substitution of FXIII at levels <50%-60%, could be suggested.
Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.
miR-142-3p expression is predictive for severe traumatic brain injury (TBI) in trauma patients
(2020)
Background: Predictive biomarkers in biofluids are the most commonly used diagnostic method, but established markers in trauma diagnostics lack accuracy. This study investigates promisingmicroRNAs(miRNA)releasedfromaffectedtissueafterseveretraumathathavepredictive values for the effects of the injury.
Methods: A retrospective analysis of prospectively collected data and blood samples of n = 33 trauma patients (ISS≥16) is provided. Levels of miR-9-5p, -124-3p, -142-3p, -219a-5p, -338-3pand-423-3p inseverelyinjuredpatients (PT)withouttraumatic braininjury (TBI) or with severe TBI (PT + TBI) and patients with isolated TBI (isTBI) were measured within 6 h after trauma.
Results: The highest miR-423-3p expression was detected in patients with severe isTBI, followed by patients with PT + TBI, and lowest levels were found in PT patients without TBI (2−∆∆Ct,p = 0.009). ApositivecorrelationbetweenmiR-423-3plevelandincreasingAIShead (p = 0.001) and risk of mortality (RISC II, p = 0.062) in trauma patients (n = 33) was found. ROC analysis of miR-423-3p levels revealed them as statistically significant to predict the severity of brain injury in trauma patients (p = 0.006). miR-124-3p was only found in patients with severe TBI, miR-338-3p was shown in all trauma groups. miR-9-5p, miR-142-3p and miR-219a-5p could not be detected in any of the four groups. Conclusion: miR-423-3p expression is significantly elevated after isolated traumatic braininjuryandpredictableforsevereTBIinthefirsthoursaftertrauma. miR-423-3pcouldrepresent a promising new biomarker to identify severe isolated TBI.
Human macrophages infiltrating hypoxic regions alter their metabolism, because oxygen becomes limited. Increased glycolysis is one of the most common cellular adaptations to hypoxia and mostly is regulated via hypoxia-inducible factor (HIF) and RAC-alpha serine/threonine–protein kinase (Akt) signaling, which gets activated under reduced oxygen content. We noticed that micro RNA (miR)-193a-3p enhances Akt phosphorylation at threonine 308 under hypoxia. In detail, miR-193a-3p suppresses the protein abundance of phosphatase PTC7 homolog (PPTC7), which in turn increases Akt phosphorylation. Lowering PPTC7 expression by siRNA or overexpressing miR-193a-3p increases Akt phosphorylation. Vice versa, inhibition of miR-193a-3p attenuates Akt activation and prevents a subsequent increase of glycolysis under hypoxia. Excluding effects of miR-193a-3p and Akt on HIF expression, stabilization, and function, we noticed phosphorylation of 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase PFKFB3 in response to the PI3K/Akt/mTOR signaling cascade. Inhibition of PFKFB3 blocked an increased glycolytic flux under hypoxia. Apparently, miR-193a-3p balances Akt phosphorylation and dephosphorylation by affecting PPTC7 protein amount. Suppression of PPTC7 increases Akt activation and phosphorylation of PFKFB3, which culminates in higher rates of glycolysis under hypoxia.
Mitochondria have a central role in regulating a range of cellular activities and host responses upon bacterial infection. Multiple pathogens affect mitochondria dynamics and functions to influence their intracellular survival or evade host immunity. On the other side, major host responses elicited against infections are directly dependent on mitochondrial functions, thus placing mitochondria centrally in maintaining homeostasis upon infection. In this review, we summarize how different bacteria and viruses impact morphological and functional changes in host mitochondria and how this manipulation can influence microbial pathogenesis as well as the host cell metabolism and immune responses.
In Deutschland existieren nur wenige Ergebnisse aus der klinischen Forschung, die im Kontext der allgemeinmedizinischen Versorgung gewonnen wurden. Dies ist u.a. damit zu begründen, dass Forschung in der Allgemeinmedizin in den Praxisalltag eingebunden sein muss, worauf die gegenwärtige Versorgungsstruktur nicht ausgelegt ist. Damit für Hausärztinnen und Hausärzte Forschung im Praxisalltag möglich ist, müssen also Strukturen geschaffen werden, die Forschung ermöglichen. Eine solche Struktur bieten Forschungspraxennetze (FPN) wie beispielsweise das Forschungspraxennetz „ForN“. ForN wurde vom Institut für Allgemeinmedizin der Goethe-Universität Frankfurt am Main im Jahr 2011 initiiert. In ForN tätige Forschungspraxen können sich mit einer Gültigkeit von fünf Jahren als „akademische Forschungspraxis“ akkreditieren lassen, wenn sie bestimmte Anforderungen erfüllen. Dazu gehört die Teilnahme an für Forschungsprojekte qualifizierenden Fortbildungen oder an Netz-begleitenden Treffen für Ärztinnen, Ärzte und Medizinische Fachangestellte (MFA) sowie die regelmäßige Teilnahme an Forschungsprojekten. Eine Verlängerung der Akkreditierung nach fünf Jahren ist möglich. Bisherige Publikationen über Forschung zu FPN bieten insbesondere Einblicke in Faktoren, welche den Beitritt in ein FPN fördern oder eher behindern. Forschung, die Faktoren der längerfristigen Mitwirkung der Praxen an FPN (wie Austrittsgründe, aber auch Motivation, eine Re-Akkreditierung anzustreben) untersucht, ist im Gegensatz dazu rar.
Diese Dissertation untersucht diese Faktoren anhand der folgenden Fragen: warum traten Forschungspraxen im Laufe der Zeit aus dem FPN ForN aus (ehemalige Mitglieder) und warum strebten andere eine Re-Akkreditierung an (aktive Mitglieder)? Weitere Unterfragen dieser Arbeit sind: welche Faktoren motivierten oder erschwerten hausärztlichen Teams eine Mitwirkung als Forschungspraxis? Wie wurde die bisherige Zusammenarbeit gesehen und als wie gut machbar wurden die Anforderungen zur Erlangung der Bezeichnung „akademische Forschungspraxis“ eingeschätzt?
Es wurde ein Fragebogen für ehemalige ärztliche ForN-Mitglieder entworfen und eingesetzt. Des Weiteren wurden Daten aus Fragebogenerhebungen von aktiven ForN-Mitgliedern (Hausärztinnen, Hausärzte, MFA) aufbereitetet. Die Daten wurden mithilfe der Datenanalysesoftware SPSS deskriptiv unter Angabe der Häufigkeiten, Mittelwerte, Standardabweichungen und Spannweiten ausgewertet.
Es konnten 14 Fragebögen von ehemaligen ärztlichen ForN-Mitgliedern aus 13 Praxen analysiert werden. Von den aktiven ForN-Mitgliedern wurden Fragebögen von 48 Ärztinnen und Ärzten sowie 57 MFA aus 41 Praxen ausgewertet. Als Gründe für den Austritt wurde von ehemaligen Mitgliedern insbesondere Zeitmangel und eine hohe Arbeitsbelastung angegeben. Weitere erschwerende Umstände waren ein Mangel an MFA, eine große Entfernung zum Standort des Instituts für Allgemeinmedizin und persönliche Gründe. Bis auf letztere Angabe waren dies auch die größten Hürden während der Teilnahme, die von aktiven Mitgliedern beschrieben wurden. Einen Beitrag für die Allgemeinmedizin zu leisten, persönliche Kompetenzen und Kompetenzen des Praxisteams zu erweitern sowie Abwechslung im Praxisalltag zu erleben, waren die wichtigsten motivierenden Faktoren einer Teilnahme im Forschungspraxennetz ForN, sowohl für aktive als auch für ehemalige Mitglieder. Die Anforderungen des FPN ForN an die Akkreditierung als Forschungspraxis wurden aus Perspektive der aktiven Mitglieder überwiegend als machbar empfunden. Nur die Umsetzung zusätzlicher, nicht verpflichtender Aktivitäten wurde als schwieriger erfüllbar bewertet. Bezüglich der Zusammenarbeit mit dem Institut für Allgemeinmedizin gaben ehemalige ärztliche ForN-Mitglieder an, eine gute Vorbereitung auf Forschungsaufgaben erfahren zu haben, dem ForN-Team des Instituts für Allgemeinmedizin vertrauen zu können und dass die Kommunikation allgemein gut war.
Gründe für den Austritt aus dem FPN und Hindernisse während der Teilnahme in ForN waren überwiegend externe Faktoren wie Zeitmangel, eine hohe Arbeitsbelastung oder ein Mangel an MFA. Auf diese Faktoren hat das Institut für Allgemeinmedizin keinen direkten Einfluss, es kann lediglich die administrativen Aufgaben innerhalb eines Forschungsprojektes für das Team einer Forschungspraxis so vorstrukturieren, dass sie möglichst gut im Praxisalltag bewältigt werden können. Ein Vergleich mit Publikationen, die sich mit förderlichen und hinderlichen Faktoren eines Beitritts in ein FPN auseinandersetzen, zeigt, dass ähnliche Gründe auch die längerfristige Mitwirkung beeinflussen.
Es ist wissenschaftlich belegt, dass hohe Feinstaubbelastungen direkt mit gesundheitlichen Schäden vergesellschaftet sind. Insbesondere in Städten, in denen die Konzentrationen besonders hoch sind, stellt dies ein ernstzunehmendes Problem dar . Ein wesentlicher Beitrag der Feinstaubbelastung ist auf anthropogene Prozesse und insbesondere auf verkehrsbedingte Emissionen zurückzuführen. Hierbei sind Abgase aus Verbrennungsmotoren sowie Brems- und Reifenabrieb zu nennen.
Für die gesundheitliche Risikobewertung einzelner Verkehrsteilnehmer werden mehrheitlich Daten stationärer Messnetzwerke herangezogen. Die Aussagekraft dieser Daten für diesen Zweck wird jedoch mitunter kritisch bewertet.
Um die Feinstaubexposition gegenüber einem Fahrzeuginsassen realistischer beurteilen zu können, erfolgt die Messung der Feinstaubkonzentration im Innenraum eines PKW mit einem mobilen Aerosolspektrometer und GPS-gestützter Standortbestimmung in Frankfurt am Main. Hierbei werden die Konzentrationen für die Partikelfraktionen PM10, PM2,5, PM1 und PMcoarse unter unterschiedlichen Bedingungen kontinuierlich gemessen.
Neben einem Vergleich der mobil gemessenen Feinstaubkonzentrationen mit Daten des stationären Messnetzwerks, werden auch lokale Konzentrationsvariationen sowie Expositionsdifferenzen zwischen Fahrten mit geöffnetem und geschlossenem Fenster analysiert.
Um einzelne Feinstaubquellen identifizieren zu können, wird die Fahrzeugumgebung mit einer Videokamera überwacht.
Im Fahrzeuginnenraum wurden Spitzenkonzentrationen von 508 µg m-3 für PM10, 133,9 µg m-3 für PM2,5, 122,9 µg m-3 für PM1 und 109,8 µg m-3 für PMcoarse (2,5-10 µm) erreicht. Die Konzentration und die Partikelgrößenverteilung im Fahrzeuginnenraum waren stark von der Umgebungsluft abhängig. Die Konzentration feinerer Partikel (PM2,5, PM1) zeigte nur geringe Schwankungen innerhalb der Stadt mit einigen signifikanten Spitzen in der Innenstadt und auf stark befahrenen Straßen. Im Gegensatz dazu wies die PMcoarse-Konzentration starke Schwankungen auf. Die höchsten Werte wurden ebenfalls in der Innenstadt und auf stark befahrenen Straßen gemessen. Durch Analyse des Videomaterials war es möglich, einige Konzentrationsspitzen mit ihren charakteristischen Partikelgrößenspektren den jeweiligen Quellen zu zuordnen.
Die absoluten Partikelkonzentrationen unterschieden sich signifikant zwischen den mobilen und den stationären Messungen, obwohl für feinere Partikel gute Korrelationen beobachtet werden konnten. Insbesondere die bei geöffnetem Fenster gemessenen Fahrzeuginnenraumkonzentrationen waren für alle Partikelgrößen deutlich höher als die entsprechenden Werte der Messstation. Schließlich erfolgte ein direkter Vergleich der Feinstaubkonzentrationen im Fahrzeug zwischen Fahrten mit geöffnetem und geschlossenem Fenster. An jedem Tag wurde nach dem Schließen des Fensters eine Konzentrationsreduktion bei allen Größenfraktionen beobachtet. Grobe Partikel (2,5-10 µm) wurden um 87,9 bis 97,4 %, feinere Partikel (1-2,5 µm) um 77,9 bis 88,2 % und die feinsten Partikel (<1 µm) um 13 bis 52 % reduziert.
Nach Interpretation der vorliegenden Daten sind mobile Messungen für eine Risikoanalyse der Feinstaubexposition auf einen Fahrzeuginsassen unerlässlich. Die Partikelkonzentration im PKW war über alle Fahrten hinweg mäßig bis hoch, wobei die Konzentration von groben Partikeln leicht durch Schließen des Fensters und Nutzung der Klimaanlage reduziert werden konnte. Die Konzentration der feineren Partikel konnte durch diese Maßnahmen nur geringfügig herabgesetzt werden.
Basierend auf den vorliegenden Ergebnissen sollte in zukünftigen Studien auf die Quantifizierung ultrafeiner Partikel eingegangen werden sowie eine ergänzende chemische Analyse der Partikel erfolgen.
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.
While the liver, specifically hepatocytes, are widely accepted as the main source for hepatitis C virus (HCV) production, the role of the liver/hepatocytes in the clearance of circulating HCV remains largely unknown. Here we evaluated the function of the liver/hepatocytes in clearing virus from the circulation by investigating viral clearance during liver transplantation and from culture medium in vitro. Frequent HCV kinetic data during liver transplantation were recorded from 5 individuals throughout the anhepatic (AH) phase and for 4 hours after reperfusion (RP), along with recordings of fluid balances. Using mathematical modeling, the serum viral clearance rate, c, was estimated. Analogously, we monitored the clearance rate of HCV at 37°C from culture medium in vitro in the absence and presence of chronically infected Huh7 human hepatoma cells. During the AH phase, in 3 transplant cases viral levels remained at pre-AH levels, while in the other 2 cases HCV declined (half-life, t1/2~1h). Immediately post-RP, virus declined in a biphasic manner in Cases 1-4 consisting of an extremely rapid (median t1/2=5min) decline followed by a slower decline (HCV t1/2=67min). In Case 5, HCV remained at the same level post-RP as at the end of AH. Declines in virus level were not explained by adjusting for dilution from IV fluid and blood products. Consistent with what was observed in the majority of patients in the anhepatic phase, the t1/2 of HCV in cell culture was much longer in the absence of chronically HCV-infected Huh7 cells. Therefore, kinetic and modeling results from both in vivo liver transplantation cases and in vitro cell culture studies suggest that the liver plays a major role in clearing HCV from the circulation.
While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from five liver transplant patients throughout the anhepatic (absence of liver) phase and for 4 hr post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n = 3) or declined (n = 2) with t1/2~1 hr. Immediately post-reperfusion, virus declined in a biphasic manner in four patients consisting of a rapid decline (t1/2 = 5 min) followed by a slower decline (t1/2 = 67 min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.
The thymus hosts the development of a specific type of adaptive immune cells called T cells. T cells orchestrate the adaptive immune response through recognition of antigen by the highly variable T-cell receptor (TCR). T-cell development is a tightly coordinated process comprising lineage commitment, somatic recombination of Tcr gene loci and selection for functional, but non-self-reactive TCRs, all interspersed with massive proliferation and cell death. Thus, the thymus produces a pool of T cells throughout life capable of responding to virtually any exogenous attack while preserving the body through self-tolerance. The thymus has been of considerable interest to both immunologists and theoretical biologists due to its multi-scale quantitative properties, bridging molecular binding, population dynamics and polyclonal repertoire specificity. Here, we review experimental strategies aimed at revealing quantitative and dynamic properties of T-cell development and how they have been implemented in mathematical modeling strategies that were reported to help understand the flexible dynamics of the highly dividing and dying thymic cell populations. Furthermore, we summarize the current challenges to estimating in vivo cellular dynamics and to reaching a next- generation multi-scale picture of T-cell development.
Neuronal hyperexcitability is a feature of Alzheimer’s disease (AD). Three main mechanisms have been proposed to explain it: i), dendritic degeneration leading to increased input resistance, ii), ion channel changes leading to enhanced intrinsic excitability, and iii), synaptic changes leading to excitation-inhibition (E/I) imbalance. However, the relative contribution of these mechanisms is not fully understood. Therefore, we performed biophysically realistic multi-compartmental modelling of excitability in reconstructed CA1 pyramidal neurons of wild-type and APP/PS1 mice, a well-established animal model of AD. We show that, for synaptic activation, the excitability promoting effects of dendritic degeneration are cancelled out by excitability decreasing effects of synaptic loss. We find an interesting balance of excitability regulation with enhanced degeneration in the basal dendrites of APP/PS1 cells potentially leading to increased excitation by the apical but decreased excitation by the basal Schaffer collateral pathway. Furthermore, our simulations reveal that three additional pathomechanistic scenarios can account for the experimentally observed increase in firing and bursting of CA1 pyramidal neurons in APP/PS1 mice. Scenario 1: increased excitatory burst input; scenario 2: enhanced E/I ratio and scenario 3: alteration of intrinsic ion channels (IAHP down-regulated; INap, INa and ICaT up-regulated) in addition to enhanced E/I ratio. Our work supports the hypothesis that pathological network and ion channel changes are major contributors to neuronal hyperexcitability in AD. Overall, our results are in line with the concept of multi-causality and degeneracy according to which multiple different disruptions are separately sufficient but no single disruption is necessary for neuronal hyperexcitability.
Polo-like kinases (PLKs) belong to a five-membered family of highly conserved serine/threonine kinases (PLK1-5) that play differentiated and essential roles as key mitotic kinases and cell cycle regulators and with this in proliferation and cellular growth. Besides, evidence is accumulating for complex and vital non-mitotic functions of PLKs. Dysregulation of PLKs is widely associated with tumorigenesis and by this, PLKs have gained increasing significance as attractive targets in cancer with diagnostic, prognostic and therapeutic potential. PLK1 has proved to have strong clinical relevance as it was found to be over-expressed in different cancer types and linked to poor patient prognosis. Targeting the diverse functions of PLKs (tumor suppressor, oncogenic) are currently at the center of numerous investigations in particular with the inhibition of PLK1 and PLK4, respectively in multiple cancer trials. Functions of PLKs and the effects of their inhibition have been extensively studied in cancer cell culture models but information is rare on how these drugs affect benign tissues and organs. As a step further towards clinical application as cancer targets, mouse models therefore play a central role. Modelling PLK function in animal models, e.g., by gene disruption or by treatment with small molecule PLK inhibitors offers promising possibilities to unveil the biological significance of PLKs in cancer maintenance and progression and give important information on PLKs’ applicability as cancer targets. In this review we aim at summarizing the approaches of modelling PLK function in mice so far with a special glimpse on the significance of PLKs in ovarian cancer and of orthotopic cancer models used in this fatal malignancy.
The NAD analogue [3-(3-acetylpyridinio)-propyl] adenosine pyrophosphate forms enzymically inactive complexes with glyceraldehyde-3-phosphate dehydrogenase from yeast and rabbit skeletal muscle. In the latter enzyme four mol of the analogue are bound with equal affinity inhibiting the enzyme in a competitive way: KI = 0.3 mM as compared to the dissociation constant KD=O.6 mм.
The brominated derivative [3- (3-bromoacetylpyridinio) -propyl] adenosine pyrophosphate is covalently bound to both enzymes causing irreversible loss of enzymic activity. Complete inactivation of the enzyme from muscle requires two moles of the analogue per mol of tetramer. The remaining two sites are still able to bind two mol of NAD+ without regain of enzymic activity. In the case of the yeast enzyme four mol of the analogue are bound. Inactivation of the rabbit muscle enzyme is accompanied by the disappearance of two out of four highly reactive sulfhydryl groups; in the yeast enzyme the four active site cysteine residues are still able to react with DTNB1 the reactivity being diminished significantly.
Hybrid formation between the native enzymes from yeast and skeletal muscle is not affected by the modification of the enzyme. Similarly the sedimentation properties of the covalently modified enzyme are indistinguishable from those of the native molecule. This indicates that both the native and the irreversibly inhibited enzyme are identical regarding their quaternary structure.
Abstract
Rheumatoid arthritis (RA) is associated with systemic osteoporosis, which leads to severe disability and low quality of life. Current therapies target osteoclasts to reduce bone degradation, but more treatment options would be required to promote bone protection by acting directly on osteoblasts (OB). Recently, the local production of dopamine in inflamed joints of RA has been observed. Thus, in this project, we aimed to determine the implication of the neurotransmitter dopamine in the bone formation process in RA. Dopamine receptors (DR) in the human bone tissue of RA or osteoarthritis (OA) patients were examined by immunohistochemistry. DR in isolated human osteoblasts (OB) was analyzed by flow cytometry, and dopamine content was evaluated by ELISA. Osteoclasts (OC) were differentiated from the PBMCs of healthy controls (HC) and RA patients. Isolated cells were treated with specific dopamine agonists. The effect of dopamine on mineralization was evaluated by Alizarin red staining. Cytokine release in supernatants was measured by ELISA. Osteoclastogenesis was evaluated with TRAP staining. OC markers were analyzed via real-time PCR and bone resorption via staining of resorption pits with toluidine blue. All DR were observed in bone tissue, especially in the bone remodeling area. Isolated OB maintained DR expression, which allowed their study in vitro. Isolated OB expressed tyrosine hydroxylase, the rate-limiting enzyme for dopamine production, and contained dopamine. The activation of D2-like DR significantly increased bone mineralization in RA osteoblasts and increased osteoclastogenesis but did not alter the expression of OC markers nor bone resorption. DR were found in the bone remodeling area of human bone tissue and dopamine can be produced by osteoblasts themselves, thus suggesting a local autocrine/paracrine pathway of dopamine in the bone. D2-like DRs are responsible for bone mineralization in osteoblasts from RA patients without an increase in bone resorption, thus suggesting the D2-like DR pathway as a possible future therapeutic target to counteract bone resorption in arthritis
Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.
Highly sensitive qualitative and quantitative automatednucleic acid amplification tests (NATs) that are commercially available for the detection of hepatitis B virus (HBV)infection have been developed only in the last few years.The potential indications for HBV NATs are: follow-up ofchronic hepatitis B, therapy and antiviral resistance monitoring, determination of infectivity and transmission risk,detection of occult (HBsAg-negative and HBV DNA-positive) infection and mutant virus which may escape serologic diagnosis, blood donor screening, and resolution ofunusual or discordant serologic constellations. Although NATs are now widely implemented in the routine diagnosis of clinical laboratories, there are several importantissues which need to be further investigated. Standardisation of NATs used for the monitoring of antiviral therapyand follow-up of chronic infection is still lacking, and theclinical significance of HBV DNA levels needs to be clarified. The influence of genetic variability in terms of genotype variation has been poorly investigated so far.Although there are highly sensitive automated NATs forblood donor screening available, their implementation is still subject to discussion and certain countries rejectedHBV DNA testing for blood donation for reasons of poor cost-effectiveness.
Uterine cervical cancer is one of the leading causes of cancer-related mortality in women worldwide. Each year, over half a million new cases are estimated, resulting in more than 300,000 deaths. While less-invasive, fertility-preserving surgical procedures can be offered to women in early stages, treatment for locally advanced disease may include radical hysterectomy, primary chemoradiotherapy (CRT) or a combination of these modalities. Concurrent platinum-based chemoradiotherapy regimens remain the first-line treatments for locally advanced cervical cancer. Despite achievements such as the introduction of angiogenesis inhibitors, and more recently immunotherapies, the overall survival of women with persistent, recurrent or metastatic disease has not been extended significantly in the last decades. Furthermore, a broad spectrum of molecular markers to predict therapy response and survival and to identify patients with high- and low-risk constellations is missing. Implementation of these markers, however, may help to further improve treatment and to develop new targeted therapies. This review aims to provide comprehensive insights into the complex mechanisms of cervical cancer pathogenesis within the context of molecular markers for predicting treatment response and prognosis.
KCNQ1 encodes the voltage-gated potassium (Kv) channel KCNQ1, also known as KvLQT1 or Kv7.1. Together with its ß-subunit KCNE1, also denoted as minK, this channel generates the slowly activating cardiac delayed rectifier current IKs, which is a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function mutations in KCNQ1 cause congenital long QT1 (LQT1) syndrome, characterized by a delayed cardiac repolarization and a prolonged QT interval in the surface electrocardiogram. Autosomal dominant loss-of-function mutations in KCNQ1 result in long QT syndrome, called Romano–Ward Syndrome (RWS), while autosomal recessive mutations lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. Here, we identified a homozygous KCNQ1 mutation, c.1892_1893insC (p.P631fs*20), in a patient with an isolated LQT syndrome (LQTS) without hearing loss. Nevertheless, the inheritance trait is autosomal recessive, with heterozygous family members being asymptomatic. The results of the electrophysiological characterization of the mutant, using voltage-clamp recordings in Xenopus laevis oocytes, are in agreement with an autosomal recessive disorder, since the IKs reduction was only observed in homomeric mutants, but not in heteromeric IKs channel complexes containing wild-type channel subunits. We found that KCNE1 rescues the KCNQ1 loss-of-function in mutant IKs channel complexes when they contain wild-type KCNQ1 subunits, as found in the heterozygous state. Action potential modellings confirmed that the recessive c.1892_1893insC LQT1 mutation only affects the APD of homozygous mutation carriers. Thus, our study provides the molecular mechanism for an atypical autosomal recessive LQT trait that lacks hearing impairment.
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2,3,4,5,6,7. Earlier studies concluded that FLVCR1 may function as a haem exporter8,9,10,11,12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14,15,16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation–π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.
Background: The increasing number of cases and hospital admissions due to COVID-19 created an urgent need for rapid, reliable testing procedures for SARS-CoV-2 in Emergency Departments (ED) in order to effectively manage hospital resources, allocate beds and prevent nosocomial spread of infection. The ID NOW™ COVID-19 assay is a simple, user-friendly, rapid molecular test run on an instrument with a small footprint enabling point-of-care diagnostics.
Methods: In the first wave, outsourced RT-PCR testing regularly required 36-48 hours before results were available. This prospective study was conducted in the second wave (October 2020-April 2021) and evaluated the impact the implementation of the ID NOW™ COVID-19 test in the ED had on clinical care processes and patient pathways. 710 patients were recruited upon arrival at the ED which included those presenting clinical symptoms, asymptomatic individuals or persons fulfilling epidemiological criteria. The first anterior nasal swab was taken by trained nurses in the ambulance or a separate consultation room. The ID NOW™ COVID-19 test was performed in the ED in strict compliance with the manufacturer’s instructions and positive or suspected cases were additionally tested with RT_PCR (cobas SARS-COV-2 RT-PCR, Roche) following collection of a second nasopharyngeal NP specimen.
Results: Swabs directly tested with the ID NOW™ COVID-19 test showed a diagnostic concordance of 98 % (sensitivity 99.59 %, specificity 94.55 %, PPV 97.6 %, NPV 99.05 %) compared to RT-PCR as reference. The 488 patients that tested positive with the ID NOW™ COVID-19 had a Ct range in RT-PCR results between 7.94 to 37.42 (in 23.2 % > 30). Two false negative results (0.28%) were recorded from patients with Ct values > 30. 14 (1.69%) discordant results were reviewed case-by-case and usually associated with either very early or very advanced stages of infection. Furthermore, patients initially negative with the ID NOW™ COVID-19 test and admitted to the hospital were tested again on days 5 and 12: no patient became positive.
Discussion: The ID NOW™ COVID-19 test for detection of SARS-CoV-2 demonstrated excellent diagnostic agreement with RT-PCR under the above-mentioned patients pathways implemented during the second wave. The main advantage of the system was the provision of reliable results within a few minutes. This not only allowed immediate initiative of appropriate therapy and care for COVID-19 (patient benefit) but provided essential information on isolation and thus available beds. This drastically helped the overall finances of the department and additionally allowed more patients to be admitted including those requiring immediate attention; this was not possible during the first wave since beds were blocked waiting for diagnostic confirmation. Our findings also show that when interpreting the results, the clinical condition and epidemiological history of the patient must be taken into account, as with any test procedure. Overall, the ID NOW™ COVID-19 test for SARS-CoV-2 provided a rapid and reliable alternative to laboratory-based RT-PCR in the real clinical setting which became an acceptable part of the daily routine within the ED and demonstrated that early patient management can mitigate the impact of the pandemic on the hospital.
Molecular surveillance of carbapenem-resistant gram-negative bacteria in liver transplant candidates
(2021)
Background: Carbapenem-resistant Gram-negative bacteria (CRGN) cause life-threatening infections due to limited antimicrobial treatment options. The occurrence of CRGN is often linked to hospitalization and antimicrobial treatment but remains incompletely understood. CRGN are common in patients with severe illness (e.g., liver transplantation patients). Using whole-genome sequencing (WGS), we aimed to elucidate the evolution of CRGN in this vulnerable cohort and to reconstruct potential transmission routes.
Methods: From 351 patients evaluated for liver transplantation, 18 CRGN isolates (from 17 patients) were analyzed. Using WGS and bioinformatic analysis, genotypes and phylogenetic relationships were explored. Potential epidemiological links were assessed by analysis of patient charts.
Results: Carbapenem-resistant (CR) Klebsiella pneumoniae (n=9) and CR Pseudomonas aeruginosa (n=7) were the predominating pathogens. In silico analysis revealed that 14/18 CRGN did not harbor carbapenemase-coding genes, whereas in 4/18 CRGN, carbapenemases (VIM-1, VIM-2, OXA-232, and OXA-72) were detected. Among all isolates, there was no evidence of plasmid transfer-mediated carbapenem resistance. A close phylogenetic relatedness was found for three K. pneumoniae isolates. Although no epidemiological context was comprehensible for the CRGN isolates, evidence was found that the isolates resulted of a transmission of a carbapenem-susceptible ancestor before individual radiation into CRGN.
Conclusion: The integrative epidemiological study reveals a high diversity of CRGN in liver cirrhosis patients. Mutation of carbapenem-susceptible ancestors appears to be the dominant way of CR acquisition rather than in-hospital transmission of CRGN or carbapenemase-encoding genetic elements. This study underlines the need to avoid transmission of carbapenem-susceptible ancestors in vulnerable patient cohorts.
Hepatic cells are sensitive to internal and external signals. Ethanol is one of the oldest and most widely used drugs in the world. The focus on the mechanistic engine of the alcohol-induced injury has been in the liver, which is responsible for the pathways of alcohol metabolism. Ethanol undergoes a phase I type of reaction, mainly catalyzed by the cytoplasmic enzyme, alcohol dehydrogenase (ADH), and by the microsomal ethanol-oxidizing system (MEOS). Reactive oxygen species (ROS) generated by cytochrome (CYP) 2E1 activity and MEOS contribute to ethanol-induced toxicity. We aimed to: (1) Describe the cellular, pathophysiological and clinical effects of alcohol misuse on the liver; (2) Select the biomarkers and analytical methods utilized by the clinical laboratory to assess alcohol exposure; (3) Provide therapeutic ideas to prevent/reduce alcohol-induced liver injury; (4) Provide up-to-date knowledge regarding the Corona virus and its affect on the liver; (5) Link rare diseases with alcohol consumption. The current review contributes to risk identification of patients with alcoholic, as well as non-alcoholic, liver disease and metabolic syndrome. Additional prevalence of ethnic, genetic, and viral vulnerabilities are presented.
Im Rahmen dieser Arbeit wurde die Rolle des Proteins S100B in humanen Neuroblastomzellen und primären hippokampalen Neurone der Ratte beim apoptotischen Zelltod untersucht. Hierfür wurden verschiedene zelltodinduzierende Agentien und Stresskonditionen verwendet. Für den exzitotoxischen, glutamatabhängigen Zelltod wurde eine NMDA-induzierte Zellschädigung sowie eine Hypoxieinduktion in einer Hypoxiekammer benutzt. Hier konnte für beide Apoptosemodelle und in beiden Zellarten eine signifikante Neuroprotektion in Anwesenheit von S100B gezeigt werden. Besonders in Hinblick auf bereits gezeigte aktive Sezernierung von S100B nach metabolomischem Stress in Astrozyten sollten die weiteren Signalwege und Effekte dieses Proteins erforscht werden. Im Zuge der Untersuchung eines möglichen Wirkungsmechanismus von S100B zeigte sich zunächst eine signifikante Aktivierung des Zellrezeptors RAGE. Weiterhin zeigte sich in primären hippokampalen Neuronen eine Aktivierung des RAF/MEK/MAPKERK-Signalwegs zumindest partiell verantwortlich für die Vermittlung der neuoprotektiven Wirkung von S100B bei NMDA-induzierter Apoptose. Durch Experimente unserer Arbeitsgruppe wurde bereits zuvor eine S100B abhängige Aktivierung von NFκB beobachtet. In dieser Arbeit konnte mit VEGF ein evtl. NFκB-abhängig aktiviertes Zielgen für die neuroprotektive Wirkung von S100B bei hypoxieinduzierter Apotpose gefunden werden. Demnach erklärt sich ein möglicher neuroprotektiver Wirkmechanismus von S100B beim exzitotoxischen Zelltod durch Aktivierung des Rezeptors RAGE an der Zelloberfläche, mit anschließender Aktivierung des MEK-Erk Signalwegs. Dieses kann seinerseits zu einer Aktivierung von NFκB in der Zelle mit Hochregulierung des VEGF-Gens führen.
Ein weiteres untersuchtes Apoptosemodell für die Rolle von S100B war die direkte DNA-Schädigung durch UV-Bestrahlung und Etoposid sowie die Schädigung durch den Proteasom-Inhibitor und p53 Aktivator Epoxomicin in humanen SHSY5Y Neuroblastoma-Zellen und primären hippokampalen Neuronen der Ratte. Auch hier zeigte sich in allen drei Modellen eine signifikante Neuroprotektion in Anwesenheit von S100B.
Da es einige Hinweise (unter anderem noch nicht publizierte Daten unserer eigenen Arbeitsgruppe) für eine Aufnahme von S100B in die Zelle gibt, wurde eine evtl. Wechselwirkung von S100B mit dem, nach DNA-Schädigung hochreguliertem, apoptoseinduzierenden Protein p53 untersucht. Hier zeigte sich, dass S100B sowohl nach DNA-Schädigung durch UV-Bestrahlung, als auch nach Etoposid-Behandlung die Hochregulierung von p53 auf Proteinebene signifikant reduziert und eine Translokation zum Zellkern verhindert. In Zusammenschau dieser Daten und den aktuellen Literaturdaten über direkte Wechselwirkungen von S100B und p53 kann man davon ausgehen, dass S100B seine Wirkung nicht nur über den Zelloberflächenrezeptor RAGE ausübt, sondern nach einem noch nicht vollständig erforschten Aufnahmemechanismus in die Zelle durch direkte Proteininteraktionen, z. B. wie hier mit dem Protein p53, in den Zellprozess insbesondere im Apoptoseprozess eingreift. Abgesehen von der in dieser Arbeit beschriebenen Herunterregulierung des p53-Proteinlevels in Anwesenheit von S100B, welche die Folge einer proteasomalen Degradation nach Formationsänderung sein kann, sollten die weiteren p53-abhängigen Apoptoseinduktionswege wie eine Veränderung von dessen Transkriptionsaktiviät, Hemmung proapoptotischer Proteine und ein evtl. Einfluss auf die Translokation von sog. Todesrezeptoren an die Zellmembran in Anwesenheit von S100B als evtl. Ursachen des neuroprotektiven Effekts von S100B weiter erforscht werden.
Im Rahmen dieser Arbeit bereits durchgeführte Untersuchungen auf Veränderungen der Expressionsrate von möglichen p53-Zielgenen haben noch keine endgültigen Ergebnisse geliefert. Zum einen ist evtl. die Auswahl der ausgewählten Zielgene nicht ausreichend gewesen und zum anderen besteht eine evtl. Limitation der semiquantitativen RT-PCR Methode gegenüber neueren Methoden wie die quantitative Real-Time-PCR in der Detektion auch kleinerer Expressionsunterschiede (siehe oben). Der Mechanismus der Neuroprotektion kann in diesem Modell abschließend noch nicht vollständig geklärt werden. Weiterführende Untersuchungen sollten den genauen Aufnahmemechanismus von S100B in die Zelle untersuchen, und die neuroprotektiven Schritte nach einer Blockierung/Herunterregulierung von p53 weiter klären.
Schätzungen der WHO zufolge waren 2015 weltweit rund 71 Millionen Menschen von einer chronischen Hepatitis C-Infektion betroffen. Die chronische Hepatitis C ist mit einem erhöhten Risiko für die Entstehung einer Leberzirrhose und eines hepatozellulären Karzinoms assoziiert. Die NS3/4A-Protease als zentraler Bestandteil der Replikationsmaschinerie des Virus spaltet das HCV-Polyprotein und ist in die Inaktivierung antiviraler Proteine involviert. Durch ihren maßgeblichen Einfluss auf die virale Fitness stellt sie einen entscheidenden Faktor für die chronische Persistenz des Virus im Wirtsorganismus dar. Die Protease ist auch eine wichtige Zielstruktur für spezifische antivirale Medikamente in der Behandlung der chronischen Hepatitis C. Der natürlich vorkommende Polymorphismus Q80K in der NS3/4A-Protease ist bei bis zu 47 % der Patienten schon vor Therapiebeginn feststellbar, insbesondere beim Genotyp 1a. Q80K führt zum Therapieversagen bei makrozyklischen Proteaseinhibitoren, insbesondere Simeprevir. Phylogenetische Analysen konnten zeigen, dass 96 % aller HCV-Gensequenzen mit Q80K von einem gemeinsamen, genetischen Vorfahren abstammen und sich die Mutation seit Mitte des 20. Jahrhunderts scheinbar stabil ausgehend vom nordamerikanischen Kontinent etabliert hat. Daneben wurden mit A91S/T und S174N sogenannte second site-Austausche identifiziert, die assoziiert mit Q80K vorkommen. Ziel dieser Arbeit war es herauszufinden, welchen Einfluss diese second site-Austausche auf die Enzymaktivität und Proteinfaltung der Protease haben und ob sie mögliche Veränderungen durch den Q80K-Polymorphismus kompensieren. Nach Expression und Aufreinigung der NS3/4A-Protease wurden die Effekte von Q80K, A91S/T und S174N auf die Enzymaktivität und Thermostabilität mittels fluoreszenzbasierter Verfahren untersucht und im Zusammenhang mit einer in silico-3D-Strukturanalyse der Protease interpretiert. Es zeigte sich, dass A91S/T und S174N jeweils zu einer Angleichung der Thermostabilität des Proteins an den Wildtyp führen und somit Defizite in der Faltung der Protease durch Q80K kompensiert werden. Aufgrund der experimentellen Daten und der Topografie dieser Austausche innerhalb der NS3-Protease-Helikase-Struktur ist von indirekten Effekten der second site-Austausche auf die replikative Fitness der Virusvarianten auszugehen. Die hier charakterisierten Austausche in der NS3/4A-Protease tragen durch eine Stabilisierung der Proteinfaltung kritisch zur Stabilität des Q80K-Polymorphismus im Proteasegen des HCV Genotyp 1a bei.
Das Verständnis von Tumorerkrankungen wurde durch neue technologische und ökonomische Verbesserungen für die Next-Generation-Sequencing Analyse (NGS) gefördert. Die Komplexität der Interpretation genomischer Daten erschwert jedoch die Anwendung von NGS-Analysen im klinischen Kontext. Die Herausforderung besteht darin bei stetig wachsendem medizinischem Wissen dieses im klinischen Kontext zu interpretieren und eine personalisierte Therapieempfehlung abzugeben. Einen ressourcensparenden Ansatz können KI-unterstützende Software-Programme bieten, welche die genomischen Varianten mit der aktuellen Literatur vergleichen, eine Bewertung der Therapieoptionen geben und klinische Studien empfehlen können. In dieser retrospektiven Arbeit wurden Patient:innen mit metastasiertem Brustkrebs (n=77) mittels gezielter NGS-Analyse anhand von sogenannten Genpanels mit 126 bzw. 540 krebsrelevanten Genen im Zeitraum von 01/2019-02/2022 untersucht. Mit Hilfe von bioinformatischen Methoden wurden patientenspezifische genomische Veränderungen mit Behandlungsoptionen abgeglichen. Diese Methoden stützen sich vollständig auf öffentliche Datenbanken über somatische Varianten mit prädiktiver Evidenz für das Ansprechen auf bestimmte Medikamente. Diese Versorgungsforschung einer repräsentativen Kohorte des Universitätsklinikums Frankfurt in Kooperation mit Regionalverbund OncoNet Rhein-Main wurden systematisch ausgewertet inklusive der Bedeutung genomischer Varianten. Das OncoNet Rhein-Main ist eine Kooperation aus führenden onkologischen Zentren und Praxen im Rhein-Main-Gebiet, welche sich als Netzwerk der Aufgabe angenommen haben Patient:innen optimal therapeutisch zu versorgen. Für 51% (39/77) der Patient:innen konnte mindestens eine gezielte Therapieoption mit einem effektivem Biomarker im gleichen Tumortyp gemäß Zulassung der Europäischen Arzneimittelbehörde (EMA) gefunden werden.
Bei 12/77 (16%) wurde mindestens eine Alteration mit einem effektivem Biomarker und einer OFF-Label Therapieoption gefunden. Bei 30% der Patient:innen wurden Veränderungen in optionalen Biomarkern gefunden, welche Resistenzmechanismen erklären. Die umfassende molekulare Analyse von Patient:innen mit fortgeschrittenem Brustkrebs erlaubt die Behandlungsoption zu verbessern und ermöglicht durch die Analyse von bekannten Resistenzmarkern auch den klinischen Verlauf besser zu verstehen. Die interdisziplinäre Besprechung der Befunde im molekularem Tumorboard ist im Hinblick auf kontinuierliches Lernen aller Beteiligten sowie zur Qualitätssicherung eine entscheidende obligate Maßnahme.
Hydroxyethylstärke (HES) ist ein kolloidales Volumenersatzmittel, das zur Volumenbehandlung bei Trauma und bei Schock und zur Verbesserung der Rheologie bei Durchblutungsstörungen angewendet wird. Amylopektin, die Grundlage von HES, wird zur Veränderung der physikalischen Eigenschaften substituiert, um eine für die Infusion geeignete Lösung herstellen zu können. Ein wichtiger Begleiteffekt dieser Substitution ist, dass durch die dadurch erzeugten Störstellen der enzymatische Abbau der Volumenersatzmittel durch Serumglykosidasen minimiert wird. Die molekularen Eigenschaften der HES können anhand der Molekulargewichtsverteilung, beschrieben durch den Gewichtsmittelwert der Molmassen Mw, den Zahlenmittelwert der Molmassen Mn und die Molmasse im Peakmaximum Mp, sowie nach dem Ausmaß der Substitution beschrieben werden. Im Handel befindliche HES-Lösungen werden anhand des Gewichtsmittelwertes der Molmassen (Mw) und der molaren Substitution (MS) gekennzeichnet. Nach bisherigen Erkenntnissen zur Speicherung der HES in Organen stellten sich die Fragen, ob die Hypothese, dass HES durch lysosomale Enzyme abgebaut wird untermauert werden kann und ob es möglich ist, die Sicherheit der HES für die Anwendung am Patienten durch gezielte Verwendung bestimmter HES-Fraktionen zu verbessern. Ziel dieser Arbeit war daher, erstmals die Molekulargewichtsverteilung der nach Infusion von HES in Milz und Leber gespeicherten HES mittels Ausschluss-Chromatographie gekoppelt mit Mehrwinkel-Laser-Streulicht-Detektion zu bestimmen. Untersucht wurden drei handelsübliche HES-Präparate mit unterschiedlichem Mw und unterschiedlicher Substitution (die Bezeichnung schließt Mw (kDa) und MS ein): HES 130/0,4 und HES 200/0,5 sowie HES 450/0,7. Je acht Wistar-Ratten pro Versuchsgruppe erhielten 18 ml HES infundiert. Die Organe wurden für die Molmassenbestimmung bis zu fünfzig Tagen nach Infusion entnommen. Die Hämoglobinkonzentrationen und Hämatokritwerte bei den Blutabnahmen in den ersten 48 Stunden wurden ermittelt und gaben Aufschluss über die Hämodilution. Als wichtigstes Ergebnis wurde eine unterschiedliche Molmassenverteilung der HES aus Milz und Leber festgestellt. In der Leber werden vorwiegend niedermolekulare Anteile gespeichert. Das Mw der HES in der Leber lag direkt nach Infusion bei 89.606±8.570 (HES 450/0,7), 20.038±1.600 (HES 200/0,5) und 23.769±2.489 (HES 130/0,4). Im Verlauf der Untersuchungen stieg das Mw in der Leber bis maximal Tag 5 (HES 450/0,7) nach Infusion zwar an, fiel dann aber bei den weiteren Bestimmungen nach mehr als 5 Tagen wieder ab. Das Peakmaximum der Molmassenverteilung der HES in der Leber blieb dabei größtenteils konstant (HES 450/0,7: ~60 kDa; HES 200/0,5: ~30 kDa; HES 130/0,4: ~30 kDa). Die Molmassenverteilung der Milz wies hingegen hochmolekulare HES auf, wobei die Molmassen im Verlauf der Zeit noch zunahmen. Das Mw nach Infusion von HES 450/0,7 stieg dabei von 148.220 Da auf 229.617 Da im Mittel an. Möglicherweise erfolgt in der Milz vor allem eine Speicherung schwer zu spaltender HES. In der Leber konnte nach Infusion aller HES-Präparate und bereits unmittelbar nach Infusion HES gefunden werden. In der Milz war nur nach Infusion der hochmolekularen, hochsubstituierten HES 450/0,7 und der mittelmolekularen, mittelsubstituierten HES 200/0,5 gespeicherte HES nachzuweisen. Nach Infusion der HES 200/0,5 war dabei nur vereinzelt und erst ab einem Tag HES in der Milz auszumachen. In der Leber war die Speicherung der HES 450/0,7 ebenfalls am längsten festzustellen, während bei HES 130/0,4 die Speicherung in der Leber nur bis 3 Tage nach Infusion bestand. Der Verlauf der Molmassenverteilung in der Leber deutet auf einen intrazellulären Abbau der HES durch lysosomale Enzyme hin, während in der Milz über einen langen Zeitraum nicht gespaltene hochmolekulare HES angereichert wird. Die niedermolekulare, niedrigsubstituierte HES ist hinsichtlich der vorhersehbaren Dauer der Speicherung als besonders günstig anzusehen. In der Leber werden jedoch bei allen HES-Präparaten niedermolekulare Anteile in Konkurrenz zur renalen Elimination aufgenommen. Daher ist die wiederholte, hochdosierte Anwendung von HES bei dekompensierter Niereninsuffizienz aufgrund der Gefahr einer mechanischen Beeinträchtigung der Leber durch dort kumulierte HES stets kritisch zu betrachten.
Mongolian spots (MS) are congenital dermal conditions resulting from neural crest-derived melanocytes migration to the skin during embryogenesis. MS incidences are highly variable in different populations. Morphologically, MS present as hyperpigmented maculae of varying size and form, ranging from round spots of 1 cm in diameter to extensive discolorations covering predominantly the lower back and buttocks. Due to their coloring, which is also dependent on the skin type, MS may mimic hematoma thus posing a challenge on the physician conducting examinations of children in cases of suspected child abuse. In the present study, MS incidences and distribution, as well as skin types, were documented in a collective of 253 children examined on the basis of suspected child abuse. From these data, a classification scheme was derived to document MS and to help identify cases with a need for recurrent examination for unambiguous interpretation of initial findings alongside the main decisive factors for re-examination such as general circumstances of the initial examination (e. g., experience of the examiner, lighting conditions) and given dermatological conditions of the patient (e. g., diaper rash).
Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given.
Monoacylglycerol lipase (MGL) expressed in cancer cells influences cancer pathogenesis but the role of MGL in the tumor microenvironment (TME) is less known. Using a syngeneic tumor model with KP cells (KrasLSL-G12D/p53fl/fl; from mouse lung adenocarcinoma), we investigated whether TME-expressed MGL plays a role in tumor growth of non-small cell lung cancer (NSCLC).
In sections of human and experimental NSCLC, MGL was found in tumor cells and various cells of the TME including macrophages and stromal cells. Mice treated with the MGL inhibitor JZL184 as well as MGL knock-out (KO) mice exhibited a lower tumor burden than the controls. The reduction in tumor growth was accompanied by an increased number of CD8+ T cells and eosinophils. Naïve CD8+ T cells showed a shift toward more effector cells in MGL KOs and an increased expression of granzyme-B and interferon-γ, indicative of enhanced tumoricidal activity. 2-arachidonoyl glycerol (2-AG) was increased in tumors of MGL KO mice, and dose-dependently induced differentiation and migration of CD8+ T cells as well as migration and activation of eosinophils in vitro.
Our results suggest that next to cancer cell-derived MGL, TME cells expressing MGL are responsible for maintaining a pro-tumorigenic environment in tumors of NSCLC.
Background: To compare severe infectious complication rates after transrectal prostate biopsies between cephalosporins and fluoroquinolones for antibiotic monoprophylaxis.
Material and Methods: In the multi-institutional cohort, between November 2014 and July 2020 patients received either cefotaxime (single dose intravenously), cefpodoxime (multiple doses orally) or fluoroquinolones (multiple-doses orally or single dose intravenously) for transrectal prostate biopsy prophylaxis. Data were prospectively acquired and retrospectively analyzed. Severe infectious complications were evaluated within 30 days after biopsy. Logistic regression models predicted biopsy-related infectious complications according to antibiotic prophylaxis, application type and patient- and procedure-related risk factors.
Results: Of 793 patients, 132 (16.6%) received a single dose of intravenous cefotaxime and were compared to 119 (15%) who received multiple doses of oral cefpodoxime and 542 (68.3%) who received fluoroquinolones as monoprophylaxis. The overall incidence of severe infectious complications was 1.0% (n=8). No significant differences were observed between the three compared groups (0.8% vs. 0.8% vs. 1.1%, p=0.9). The overall rate of urosepsis was 0.3% and did not significantly differ between the three compared groups as well.
Conclusion: Monoprophylaxis with third generation cephalosporins was efficient in preventing severe infectious complications after prostate biopsy. Single intravenous dose of cefotaxime and multiday regimen of oral cefpodoxime showed a low incidence of infectious complications <1%. No differences were observed in comparison to fluoroquinolones.
Einleitung: OTSC Proctology ist ein Verfahren in der Analfistelchirurgie dessen Erfolgsaussichten auch 9 Jahre nach der ersten klinischen Anwendung nicht abschließend beurteilt werden können. Die wenigen bisher publizierten Studien zeigen sehr divergente Ergebnisse mit Heilungsraten von 10 bis 90%.
Material und Methoden: Wir führten eine retrospektive Auswertung der Behandlungsergebnisse aller konsekutiven Patienten, die in dem Zeitraum vom 01.03.2014 bis 31.03.2017 in der koloproktologischen Abteilung der DKD Helios Klinik Wiesbaden mittels OTSC-Verfahren wegen Analfisteln operiert wurden, durch. Erfasst wurden Alter, Geschlecht, OP- und Aufenthaltsdauer, Operateur, Fistelart und –lokalisation, Vorhandensein von Stoma und CED, plastischer Fistelverschluss (PFV) in der Anamnese, Clipverbleib nach der OP, Dauer des Follow-ups, Komplikationen sowie postoperative Schmerzsituation. Die Datenauswertung erfolgte mittels deskriptiver Statistik bei Subgruppenanalyse unter Verwendung der Statistiksoftware SPSS 20.
Ergebnisse: Es wurden insgesamt 68 Fälle eingeschlossen, davon 37% weiblich und 63% männlich. Das durchschnittliche Alter betrug 52 Jahre (25 – 81). 19 (28%) Patienten litten an CED, 11 (16%) Patienten hatten ein Stoma. 34 (50%) der Patienten hatten plastischen Fistelverschluss in der Anamnese. Die Verteilung nach Fisteltyp war wie folgt: 58 (85%) transsphinktär, 4 (6%) suprasphinktär, 3 (4%) intersphinktär, 1 (1,5%) rektovaginal, 1 (1,5%) rektourethral und 1 (1,5%) Pouchfistel. Die häufigsten Fistelokalisationen waren bei 6 h (N = 26, 38%), 12 h (N = 14, 21%), 7 h (N = 7, 10%) und 3h (N = 5, 7%) SSL. Die durchschnittlichen OP-Dauer und stationärer Aufenthalt betrugen 25 min (6 – 90) und 7 Tage (1 – 14 Tage) entsprechend. Die durchschnittliche Dauer des Follow-ups betrug 29 Monate (10 – 36).
Die Fistelheilung im Gesamtkollektiv lag bei 48,5%, 1 (1,5%) Patient war lost-tofollow-up. In der weiblichen (N = 25) und männlichen (N = 43) Kohorte fand die Heilung in 40% und 53% der Fälle entsprechend statt. Die Heilungsraten bei intersphinktären (N = 3), transsphinktären (N = 58) und suprasphinktären (N = 4) Fisteln lagen bei 100%, 46,5% und 50% entsprechend, eine rektovaginale und eine rektourethrale Fistel sind nicht geheilt. Eine Pouchfistel ist geheilt.
Die Heilungsraten bei 6h und 12h SSL lagen bei 58% und 14% entsprechend mit deutlichem Vorteil bei posteriorer Fistellage. Dieser Vorteil blieb nach der Aufteilung der Fisteln in anteriore (N = 30) und posteriore (N = 38), mit
Heilungsraten von 33% und 60,5% entsprechend, bestehen. In den Subgruppen ohne CED (N = 49) und mit CED (N = 19) lagen die Heilungsraten bei 53% und 35% entsprechend. In den Subgruppen ohne PFV (N = 34) und mit PFV (N = 34) lagen die Heilungsraten bei 59% und 38% entsprechend. In der Subgruppe ohne Stoma (N = 57) wurde eine Heilung in 47% der Fälle, in der Subgruppe mit Stoma (N = 11) in 55% beobachtet. In der Subgruppe mit den kryploglandulären Fisteln (N = 47) war die Heilung in 55,3% zu sehen und in der Subgruppe mit
kryptoglandulären Fisteln ohne PFV (N = 27) bei 63%.
In 48 (70,6%) Fällen wurde der Clip aktiv entfernt, in 11 (16,2%) Fällen kam es zum Spontanverlust und in 8 (11,8%) Fällen blieb der Clip in situ. Die durchschnittliche Zeit bis zur Klammerentfernung betrug 4 Monate. Die Heilungsraten bei Clipentfernung, Clipverbleib und Clipspontanabgang lagen bei 42%, 100% und 45% entsprechend.
Der maximale Schmerz nach NRS 0 – 2 bei 61% der Patienten, NRS 3 – 4 bei 28% und NRS 5 – 7 nur bei 11%. Bei 50% der Fälle war kein Opiat erforderlich und bei 39% der Fälle erfolgte die Opiateinnahme nicht länger als 2 Tage.
Die Komplikationen waren sehr selten: eine Nachblutung mit Clipdislokation (1,5%), ein Analabszess (1,5%), 2 Fälle (3%) der neuaufgetretenen Stuhlschmieren und 1 (1,5%) Wundheilungsstörung intraanal, die spontan abheilte. In 4 (6%) Fällen kam es zur Klammerdislokation vom inneren
Fistelostium mit konsekutiver Fistelpersistenz.
Fazit: OTSC ist ein komplikationsarmes und schmerzarmes Verfahren mit kurzer OP-Zeit und könnte einen festen Platz in der Analfistelchirurgie einnehmen. Die bestmöglichen Ergebnisse lassen sich bei dorsal gelegenen Analfisteln in
nichtvoroperierten Patienten ohne CED erzielen.
Background: Mesenchymal stromal cells (MSCs), multipotent progenitors that can be isolated from a variety of different tissues, are becoming increasingly important as cell therapeutics targeting immunopathologies and tissue regeneration. Current protocols for MSC isolation from bone marrow (BM) rely on density gradient centrifugation (DGC), and the production of sufficient MSC doses is a critical factor for conducting clinical MSC trials. Previously, a Good Manufacturing Practice (GMP)–compatible non-woven fabric filter device system to isolate MSCs was developed to increase the MSC yield from the BM. The aim of our study was to compare high-resolution phenotypic and functional characteristics of BM-MSCs isolated with this device and with standard DGC technology.
Methods: Human BM samples from 5 donors were analyzed. Each sample was divided equally, processing by DGC, and with the filter device. Stem cell content was assessed by quantification of colony-forming units fibroblasts (CFU-F). Immunophenotype was analyzed by multicolor flow cytometry. In vitro trilineage differentiation potential, trophic factors, and IDO-1 production were assessed. Functionally, immunomodulatory potential, wound healing, and angiogenesis were assayed in vitro.
Results: The CFU-F yield was 15-fold higher in the MSC preparations isolated with the device compared to those isolated by DGC. Consequently, the MSC yield that could be manufactured at passage 3 per mL collected BM was more than 10 times higher in the device group compared to DGC (1.65 × 109 vs. 1.45 × 108). The immunomodulatory potential and IDO-1 production showed donor-to-donor variabilities without differences between fabric filter-isolated and DGC-isolated MSCs. The results from the wound closure assays, the tube formation assays, and the trilineage differentiation assays were similar between the groups with respect to the isolation method. Sixty-four MSC subpopulations could be quantified with CD140a+CD119+CD146+ as most common phenotype group, and CD140a+CD119+CD146+MSCA-1–CD106–CD271– and CD140a+CD119+CD146–MSCA-1–CD106–CD271– as most frequent MSC subpopulations. As trophic factors hepatocyte growth factor, epidermal growth factor, brain-derived neurotrophic factor, angiopoietin-1, and vascular endothelial growth factor A could be detected in both groups with considerable variability between donors, but independent of the respective MSC isolation technique.
Conclusion: The isolation of MSCs using a GMP-compatible fabric filter system device resulted in higher yield of CFU-F, producing substantially more MSCs with similar subpopulation composition and functional characteristics as MSCs isolated by DGC.
A 24-year-old patient from Cameroon presented to our hospital because of a foreign structure in her left eye. To our knowledge, for the first time, fluorescent microscopy revealed motile microfilariae, and the diagnosis of loiasis was established. Despite substantial microfilaremia, eosinophilia only unmasked after the initiation of antiparasitic therapy.
Spinocerebellar ataxia type 2 (SCA2) is caused by polyglutamine expansion in Ataxin-2 (ATXN2). This factor binds RNA/proteins to modify metabolism after stress, and to control calcium (Ca2+) homeostasis after stimuli. Cerebellar ataxias and corticospinal motor neuron degeneration are determined by gain/loss in ATXN2 function, so we aimed to identify key molecules in this atrophic process, as potential disease progression markers. Our Atxn2-CAG100-Knock-In mouse faithfully models features observed in patients at pre-onset, early and terminal stages. Here, its cerebellar global RNA profiling revealed downregulation of signaling cascades to precede motor deficits. Validation work at mRNA/protein level defined alterations that were independent of constant physiological ATXN2 functions, but specific for RNA/aggregation toxicity, and progressive across the short lifespan. The earliest changes were detected at three months among Ca2+ channels/transporters (Itpr1, Ryr3, Atp2a2, Atp2a3, Trpc3), IP3 metabolism (Plcg1, Inpp5a, Itpka), and Ca2+-Calmodulin dependent kinases (Camk2a, Camk4). CaMKIV–Sam68 control over alternative splicing of Nrxn1, an adhesion component of glutamatergic synapses between granule and Purkinje neurons, was found to be affected. Systematic screening of pre/post-synapse components, with dendrite morphology assessment, suggested early impairment of CamKIIα abundance together with the weakening of parallel fiber connectivity. These data reveal molecular changes due to ATXN2 pathology, primarily impacting excitability and communication.
Sphingosine 1 phosphate (S1P) lyase (Sgpl1) catalyses the irreversible cleavage of S1P and thereby the last step of sphingolipid degradation. Loss of Sgpl1 in humans and mice leads to accumulation of sphingolipids and multiple organ injuries. Here, we addressed the role of hepatocyte Sgpl1 for regulation of sphingolipid homoeostasis by generating mice with hepatocyte-specific deletion of Sgpl1 (Sgpl1HepKO mice). Sgpl1HepKO mice had normal body weight, liver weight, liver structure and liver enzymes both at the age of 8 weeks and 8 months. S1P, sphingosine and ceramides, but not glucosylceramides or sphingomyelin, were elevated by ~1.5–2-fold in liver, and this phenotype did not progress with age. Several ceramides were elevated in plasma, while plasma S1P was normal. Interestingly, S1P and glucosylceramides, but not ceramides, were elevated in bile of Sgpl1HepKO mice. Furthermore, liver cholesterol was elevated, while LDL cholesterol decreased in 8-month-old mice. In agreement, the LDL receptor was upregulated, suggesting enhanced uptake of LDL cholesterol. Expression of peroxisome proliferator-activated receptor-γ, liver X receptor and fatty acid synthase was unaltered. These data show that mouse hepatocytes largely compensate the loss of Sgpl1 by secretion of accumulating sphingolipids in a specific manner into blood and bile, so that they can be excreted or degraded elsewhere.
Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.
Einleitung: Die stereotaktische Laserthermoablation (SLTA) stellt eine minimal-invasive Behandlung für therapierefraktäre Epilepsien auf dem Boden eines hypothalamischen Hamartoms (HH) dar. Durch die weitreichenden Folgen einer therapierefraktären Epilepsie können hohe direkte Kosten entstehen, die durch eine zu erzielende Anfallsfreiheit gesenkt werden können.
Methoden: Anhand einer Patientin mit einem HH sollen die Auswirkungen einer solchen Erkrankung beleuchtet und der Krankheitsverlauf nach erfolgter SLTA dargestellt werden. Zur Beurteilung der Kosteneffizienz der SLTA wurden die direkten Kosten, basierend auf den Krankenversicherungsdaten der Patientin, über die Versicherungsjahre 2017 bis 2020 analysiert.
Ergebnisse:
Bei der Patientin bestand eine hochaktive, medikamentenrefraktäre Epilepsie mit erhöhtem Verletzungsrisiko und zunehmender Verschlechterung der schulischen Leistung und der psychischen Verfassung. Begleitend bestand durch das HH eine Pubertas praecox. Nach SLTA entwickelte die Patientin mit einem Follow-up von 26 Monaten eine vollständige Anfallsfreiheit sowie eine endokrinologische Stabilisierung, sodass die antikonvulsive als auch die hormonelle Medikation im Verlauf beendet werden konnten. Relevante persistierende Komplikationen wurden nicht beobachtet. Die direkten jährlichen Kosten (stationär [ausschließlich der SLTA selbst]/ambulant/Medikamente) reduzierten sich von € 6603 in 2017 und € 12.903 in 2018 auf € 3609 in 2019 und zuletzt € 617 in 2020, was einer Reduktion von bis zu 95 % (2018 gegenüber 2020) entsprach. Zusätzlich konnten die Kosten einer geplanten Integrationsassistenz von schätzungsweise € 18.000/Jahr eingespart werden.
Schlussfolgerung: Die SLTA stellt eine effektive und risikoarme Behandlung von HH dar und führt bereits nach 2 Jahren zu einer relevanten Einsparung der direkten Kosten, was bei der Kosten-Nutzen-Abwägung der SLTA einzubeziehen ist.
Introduction: Combination therapy for melanoma brain metastases (MM) using stereotactic radiosurgery (SRS) and immune checkpoint-inhibition (ICI) or targeted therapy (TT) is currently of high interest. In this collective, time evolution and incidence of imaging findings indicative of pseudoprogression is sparsely researched. We therefore investigated time-course of MRI characteristics in these patients.
Methods: Data were obtained retrospectively from 27 patients (12 female, 15 male; mean 61 years, total of 169 MMs). Single lesion volumes, total MM burden and edema volumes were analyzed at baseline and follow-up MRIs in 2 months intervals after SRS up to 24 months. The occurrence of intralesional hemorrhages was recorded.
Results: 17 patients (80 MM) received ICI, 8 (62 MM) TT and 2 (27 MM) ICI + TT concomitantly to SRS. MM-localization was frontal (n = 89), temporal (n = 23), parietal (n = 20), occipital (n = 10), basal ganglia/thalamus/insula (n = 10) and cerebellar (n = 10). A volumetric progression of MM 2–4 months after SRS was observed in combined treatment with ICI (p = 0.028) and ICI + TT (p = 0.043), whereas MMs treated with TT showed an early volumetric regression (p = 0.004). Edema volumes moderately correlated with total MM volumes (r = 0.57; p < 0.0001). Volumetric behavior did not differ significantly over time regarding lesions’ initial sizes or localizations. No significant differences between groups were observed regarding rates of post-SRS intralesional hemorrhages.
Conclusion: Reversible volumetric increases in terms of pseudoprogression are observed 2–4 months after SRS in patients with MM concomitantly treated with ICI and ICI + TT, rarely after TT. Edema volumes mirror total MM volumes. Medical treatment type does not significantly affect rates of intralesional hemorrhage.
Die randomisierte, dreiarmig kontrollierte Studie zu täglicher, peroraler Zusatzkost (ONS) bei Hämodialysepatienten (CHD) im Endstadium der Niereninsuffizienz (ESRD) über 6 Monate zeigte keine signifikanten Verbesserungen hinsichtlich folgender Nutritions-/Retentions- und Inflammationsparameter: Subjective Global Assessment (SGA); Body Mass Index (BMI); Querschnitt des Muskulus Iliopsoas,Oberarmumfang und Dicke des Unterhautfettgewebes (MRT); örperzellmasse und Phasenwinkel (Bioimpedanzanalyse BIA); Tumornekrosefaktor α (TNFα); Interleukin 1β und 6 (IL-1β und IL-6); C-Reaktives Protein (CRP). Der Querschnitt des Muskulus biceps brachii blieb in der Kontrollgruppe anfangs und zum Ende höher wie in den Interventionsgruppen. Der Serumkreatininwert der Interventionsgruppe mit HIV war anfangs geringer als in den übrigen Gruppen, die glomeruläre Filtrationsrate entsprechend besser, zum Ende waren diese Unterschiede nivelliert. Der Hauptbefund liegt in der hohen Mortalitätsrate der HIV-positiven Hämodialysepatienten (2 von 7 Pat., 28,6%), von denen beide im SGA als schwer mangel-/fehlernährt eingestuft wurden. Die Therapie eines Malnutritions-Infalmmations-Komplexes ist nicht allein durch orale Zusatzkost möglich. Weitere Studien müssen multimodale Konzepte zur Diagnose und zur Therapie erforschen. Hierzu kann perorale Zusatzkost ein einfach durchzuführendes Mittel als Teil der Behandlungsstrategie sein, zur erweiterten Diagnose kann die Bioimpedanzanalyse eine Möglichkeit sein, um den Teilaspekt der Nutritionskontrolle zu erfüllen.
Introduction: Cancer patients tend to prefer oral instead of parenteral chemotherapy. To date, there is little evidence on the medication adherence in cancer patients. We investigated medication adherence to tyrosine kinase inhibitors in patients suffering from non-small cell lung cancer. Methods: Tyrosine kinase inhibitor adherence was measured electronically by MEMS® (medication event monitoring system) over at least six months. Adherence rates were calculated in terms of Dosing Compliance, Timing Compliance, Taking Compliance, and Drug Holidays. Patients were dichotomized as adherent when Dosing Compliance and Timing Compliance were ≥80%, Taking Compliance ranged between 90 and 110%, and <1 Drug Holiday was registered. Quality of life was assessed by two questionnaires (EORTC QLQ-C30 version 3.0, EORTC QLQ-LC13) at three time points. Adverse drug events were reported via patient diaries. Results: Out of 32 patients enrolled, data from 23 patients were evaluable. Median Dosing Compliance, Taking Compliance, and Timing Compliance adherence rates of tyrosine kinase inhibitor intake amounted to 100%, 98%, and 99%, respectively; Drug Holidays were observed in three patients. Four patients were dichotomized as non-adherent. Three of them had a twice-daily tyrosine kinase inhibitor regimen. Median quality of life scores amounted to 67 (max. 100) and remained unchanged over the study period. Fatigue and rash were the most frequently reported adverse drug events. Conclusion: Medication adherence of non-small cell lung cancer patients treated with tyrosine kinase inhibitors was extraordinarily high and is likely to support the effectiveness of tyrosine kinase inhibitor treatment and a good quality of life over a long period of time. Adherence facilitating information and education is especially relevant for patients taking tyrosine kinase inhibitors in a twice-daily regimen.
Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.
The detection of multiple biomolecule classes in one go is highly desirable for a wide variety of areas, and in particular for point-of-care diagnostics. For example, wound infections are a major problem for patient’s health and cause huge efforts in our healthcare system. In this regard, monitoring infected wounds through simultaneous detection of pathogens via nucleic acid analysis and detection of local inflammation biomarkers is key in order to enable a personalized therapy, improve the clinical outcome and thus, leading to a reduction of overall healthcare costs. In this regard, wound exudate offers an attractive sample material which can be collected in a non-invasive manner. Here, we report the development of a Multianalyte-Assay detecting inflammation biomarkers and pathogen DNA simultaneously from one sample within 35 min. Protein-compatible amplification and labeling transforms nucleic acid information into the measurement principle for protein detection. The combination with rapid detection via lateral flow immunoassay enables a fast and straightforward analysis of multiple biomolecule classes using identical assay conditions. To demonstrate the feasibility of the Multianalyte-Assay, the proinflammatory cytokine interleukin-6 (IL-6) and gDNA of the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) are used. The detection limits of 4 ng/mL IL-6 and 70 copies/reaction P. aeruginosa gDNA meet the clinically relevant range and thus, having tremendous potential to improve the wound management at the point-of-care.
Background: The diagnostic accuracy of the Elecsys® HCV Duo antigen-antibody combination immunoassay (Roche Diagnostics GmbH) was evaluated for the detection of hepatitis C virus (HCV) infection, versus commercially available comparators.
Methods: This multicenter study (August 2020–March 2021) assessed the specificity of the Elecsys HCV Duo immunoassay and comparator assays in blood donor and routine clinical laboratory samples; sensitivity was determined in confirmed HCV-positive samples and seroconversion panels. The Elecsys HCV Duo immunoassay was compared with the Monolisa HCV Ag-Ab ULTRA V2, Murex HCV Ag/Ab Combination and ARCHITECT HCV Ag assays, as well as nucleic acid testing (NAT). The antibody (anti-HCV) module of the Elecsys HCV Duo immunoassay was compared with the Elecsys Anti-HCV II, Alinity s Anti-HCV, ARCHITECT Anti-HCV and RIBA HCV 3.0 SIA assays.
Results: The specificity of the Elecsys HCV Duo immunoassay was 99.94% (95% confidence interval [CI], 99.89–99.97) and 99.92% (95% CI, 99.71–99.99) in blood donor (n = 20,634) and routine clinical laboratory samples (n = 2531), respectively. The specificity of the Elecsys HCV Duo immunoassay was similar or better than comparator assays. The sensitivity of the Elecsys HCV Duo immunoassay in confirmed HCV-positive samples (n = 257) was 99.6%. In seroconversion panels, the Elecsys HCV Duo immunoassay detected infections earlier (2.2–21.9 days) than all but one of the comparator assays and detected HCV 1.8 days later than NAT.
Conclusions: The Elecsys HCV Duo immunoassay shows high diagnostic accuracy, reduces the diagnostic window, and could be used when NAT is not possible.
Objective: This study was undertaken to quantify epilepsy-related costs of illness (COI) in Germany and identify cost-driving factors.
Methods: COI were calculated among adults with epilepsy of different etiologies and severities. Multiple regression analysis was applied to determine any epilepsy-related and sociodemographic factors that serve as cost-driving factors.
Results: In total, 486 patients were included, with a mean age of 40.5 ± 15.5 years (range = 18–83 years, 58.2% women). Mean 3-month COI were estimated at €4911, €2782, and €2598 for focal, genetic generalized, and unclassified epilepsy, respectively. The mean COI for patients with drug-refractory epilepsy (DRE; €7850) were higher than those for patients with non-DRE (€4720), patients with occasional seizures (€3596), or patients with seizures in remission for >1 year (€2409). Identified cost-driving factors for total COI included relevant disability (unstandardized regression coefficient b = €2218), poorer education (b = €2114), living alone (b = €2612), DRE (b = €1831), and frequent seizures (b = €2385). Younger age groups of 18–24 years (b = −€2945) and 25–34 years (b = −€1418) were found to have lower overall expenditures. A relevant disability (b = €441), DRE (b = €1253), frequent seizures (b = €735), and the need for specialized daycare (b = €749) were associated with higher direct COI, and poorer education (b = €1969), living alone (b = €2612), the presence of a relevant disability (b = €1809), DRE (b = €1831), and frequent seizures (b = €2385) were associated with higher indirect COI.
Significance: This analysis provides up-to-date COI data for use in further health economics analyses, highlighting the high economic impacts associated with disease severity, disability, and disease-related loss of productivity among adult patients with epilepsy. The identified cost drivers could be used as therapeutic and socioeconomic targets for future cost-containment strategies.
Patient care in a neurointensive care unit (neuro-ICU) is challenging. Multidrug-resistant organisms (MDROs) are increasingly common in the routine clinical practice. We evaluated the impact of infection with MDROs on outcomes in patients with subarachnoid hemorrhage (SAH). A single-center retrospective analysis of SAH cases involving patients treated in the neuro-ICU was performed. The outcome was assessed 6 months after SAH using the modified Rankin Scale [mRS, favorable (0–2) and unfavorable (3–6)]. Data were compared by matched-pair analysis. Patient characteristics were well matched in the MDRO (n = 61) and control (n = 61) groups. In this center, one nurse was assigned to a two-bed room. If a MDRO was detected, the patient was isolated, and the nurse was assigned to the patient infected with the MDRO. In the MDRO group, 29 patients (48%) had a favorable outcome, while 25 patients (41%) in the control group had a favorable outcome; the difference was not significant (p > 0.05). Independent prognostic factors for unfavorable outcomes were worse status at admission (OR = 3.1), concomitant intracerebral hematoma (ICH) (OR = 3.7), and delayed cerebral ischemia (DCI) (OR = 6.8). Infection with MRDOs did not have a negative impact on the outcome in SAH patients. Slightly better outcomes were observed in SAH patients infected with MDROs, suggesting the benefit of individual care.
SAMHD1 is discussed as a tumour suppressor protein, but its potential role in cancer has only been investigated in very few cancer types. Here, we performed a systematic analysis of the TCGA (adult cancer) and TARGET (paediatric cancer) databases, the results of which did not suggest that SAMHD1 should be regarded as a bona fide tumour suppressor. SAMHD1 mutations that interfere with SAMHD1 function were not associated with poor outcome, which would be expected for a tumour suppressor. High SAMHD1 tumour levels were associated with increased survival in some cancer entities and reduced survival in others. Moreover, the data suggested differences in the role of SAMHD1 between males and females and between different races. Often, there was no significant relationship between SAMHD1 levels and cancer outcome. Taken together, our results indicate that SAMHD1 may exert pro- or anti-tumourigenic effects and that SAMHD1 is involved in the oncogenic process in a minority of cancer cases. These findings seem to be in disaccord with a perception and narrative forming in the field suggesting that SAMHD1 is a tumour suppressor. A systematic literature review confirmed that most of the available scientific articles focus on a potential role of SAMHD1 as a tumour suppressor. The reasons for this remain unclear but may include confirmation bias and publication bias. Our findings emphasise that hypotheses, perceptions, and assumptions need to be continuously challenged by using all available data and evidence.
SAMHD1 is discussed as a tumour suppressor protein, but its potential role in cancer has only been investigated in very few cancer types. Here, we performed a systematic analysis of the TCGA (adult cancer) and TARGET (paediatric cancer) databases, the results of which did not suggest that SAMHD1 should be regarded as a bona fide tumour suppressor. SAMHD1 mutations that interfere with SAMHD1 function were not associated with poor outcome, which would be expected for a tumour suppressor. High SAMHD1 tumour levels were associated with increased survival in some cancer entities and reduced survival in others. Moreover, the data suggested differences in the role of SAMHD1 between males and females and between different races. Often, there was no significant relationship between SAMHD1 levels and cancer outcome. Taken together, our results indicate that SAMHD1 may exert pro-or anti-tumourigenic effects and that SAMHD1 is involved in the oncogenic process in a minority of cancer cases. These findings seem to be in disaccord with a perception and narrative forming in the field suggesting that SAMHD1 is a tumour suppressor. A systematic literature review confirmed that most of the available scientific articles focus on a potential role of SAMHD1 as a tumour suppressor. The reasons for this remain unclear but may include confirmation bias and publication bias. Our findings emphasise that hypotheses, perceptions, and assumptions need to be continuously challenged by using all available data and evidence.
Colorectal cancer (CRC) is one of the most frequently diagnosed tumor in humans and one of the most common causes of cancer-related death worldwide. The pathogenesis of CRC follows a multistage process which together with somatic gene mutations is mainly attributed to the dysregulation of signaling pathways critically involved in the maintenance of homeostasis of epithelial integrity in the intestine. A growing number of studies has highlighted the critical impact of members of the tripartite motif (TRIM) protein family on most types of human malignancies including CRC. In accordance, abundant expression of many TRIM proteins has been observed in CRC tissues and is frequently correlating with poor survival of patients. Notably, some TRIM members can act as tumor suppressors depending on the context and the type of cancer which has been assessed. Mechanistically, most cancer-related TRIMs have a critical impact on cell cycle control, apoptosis, epithelial–mesenchymal transition (EMT), metastasis, and inflammation mainly through directly interfering with diverse oncogenic signaling pathways. In addition, some recent publications have emphasized the emerging role of some TRIM members to act as transcription factors and RNA-stabilizing factors thus adding a further level of complexity to the pleiotropic biological activities of TRIM proteins. The current review focuses on oncogenic signaling processes targeted by different TRIMs and their particular role in the development of CRC. A better understanding of the crosstalk of TRIMs with these signaling pathways relevant for CRC development is an important prerequisite for the validation of TRIM proteins as novel biomarkers and as potential targets of future therapies for CRC.
Multilevel-Untersuchung des nitrinergen Systems bei affektiven Störungen und schizophrenen Psychosen
(2023)
Das nitrinerge System und damit auch NOx als Neurotransmitter werden mit der Entstehung verschiedener psychischer Erkrankungen in Verbindung gebracht. Die genaue Rolle des Botenstoffs ist jedoch nicht ausreichend geklärt und auch die Frage, ob dieser als diagnostischer oder prädiktiver Biomarker nützlich sein könnte, ist unbeantwortet. In der vorliegenden Arbeit wurde folglich untersucht, ob es Unterschiede zwischen den Diagnosegruppen MDD, BIP, SCZ und der Kontrollgruppe bezüglich peripherer NOx- Konzentrationen gibt. Darüber hinaus wurden Unterschiede innerhalb der Diagnosegruppen im Krankheitsverlauf im Sinne von Phasenunterschieden mittels zweier Messzeitpunkte untersucht und analysiert, ob es Korrelationen mit genetischen Variationen in NOS-Genabschnitten gibt. Insgesamt wurden 185 Probanden in die Studie mitaufgenommen: 52 gesunde CTRL, 43 Patienten mit MDD, 41 Patienten mit BIP und 49 Patienten mit SCZ. Biochemische, genetische und klinische Daten wurden bei Aufnahme und Entlassung in der psychiatrischen Abteilung des Universitätsklinikums Frankfurt erhoben. Klinische Daten, die den Symptomverlauf 90 und die Erkrankungsschwere beurteilten, nutzten dazu standardisierte teilstrukturierte klinische Interviews. Biochemische Daten wurden mittels im Serum gemessener NOx- Spiegel quantifiziert. Bezüglich der Untersuchung der Risikogenvarianten wurden Probanden anhand des NOS1 ex1f-VNTR-Polymorphismus sowie SNPs in den Genen NOS1, NOS3 und NOS1AP genotypisiert. Bei Aufnahme wiesen SCZ-Patienten im Vergleich zu CTRL-, MDD- und BIP-Gruppen signifikant höhere NOx- Konzentrationen auf. Während NOx- Spiegel im Behandlungsverlauf bei MDD- und BIP-Patienten signifikant zunahmen, konnte dies bei SCZ-Patienten nicht beobachtet werden. Weiterhin konnte gezeigt werden, dass Patienten, deren depressive Beschwerden nicht relevant zurückgingen, bei Entlassung signifikant höhere NOx- Konzentrationen aufwiesen, was durch die Beobachtung einer signifikant positiven Korrelation zwischen NOx- Serumspiegeln und depressiven Symptomen bei Entlassung unterstützt wurde. Bei der genetischen Untersuchung der Daten fiel auf, dass homozygote Träger des kurzen VNTR-Allels signifikant erhöhte NOx- Konzentrationen besaßen. Diese Ergebnisse blieben bei jenen Trägern auch nach Entlassung signifikant. Insgesamt gibt es Hinweise darauf, dass erhöhte periphere NOx- Metabolitkonzentrationen mit einer Zunahme der Psychopathologie bzw. der Erkrankungsschwere einhergehen könnten, was möglicherweise auf den NOS1 ex1f-VNRT-Polymorphismus zurückzuführen ist. Außerdem zeigten zwei SNPs, welche beide im NOS1AP-Gen lokalisiert sind, bei BIP Patienten signifikant gesteigerte NOx- Werte. Die vorliegenden Ergebnisse deuten darauf hin, dass NO-Signalübertragung und NOS-Genotypen in der Pathogenese psychischer Erkrankungen eine Rolle spielen könnten. Ob diese Veränderungen allerdings kausal mit Krankheitsprozessen zu tun haben oder ob es eher Epiphänomene der Erkrankungen sind, kann mit dieser Studie nicht geklärt werden. Die Genvarianten könnten wiederum bei der Regulierung von peripheren NOx- Konzentrationen von Bedeutung sein. Die Arbeit liefert zudem Hinweise, die Verwendung von NOx als möglichen peripheren Biomarker weiter zu verfeinern und zu untersuchen. Zukünftige Studien, die die Wirksamkeit von NOx- modulierenden Pharmaka untersuchen, könnten davon profitieren, Diagnosegruppen nach Subgruppen einzuteilen, die sowohl NOS Risikogenvarianten als auch periphere NOx- Spiegel im Sinne eines Biomarker beachten.
For medicine to fulfill its promise of personalized treatments based on a better understanding of disease biology, computational and statistical tools must exist to analyze the increasing amount of patient data that becomes available. A particular challenge is that several types of data are being measured to cope with the complexity of the underlying systems, enhance predictive modeling and enrich molecular understanding.
Here we review a number of recent approaches that specialize in the analysis of multimodal data in the context of predictive biomedicine. We focus on methods that combine different OMIC measurements with image or genome variation data. Our overview shows the diversity of methods that address analysis challenges and reveals new avenues for novel developments.
Spontaneous brain activity builds the foundation for human cognitive processing during external demands. Neuroimaging studies based on functional magnetic resonance imaging (fMRI) identified specific characteristics of spontaneous (intrinsic) brain dynamics to be associated with individual differences in general cognitive ability, i.e., intelligence. However, fMRI research is inherently limited by low temporal resolution, thus, preventing conclusions about neural fluctuations within the range of milliseconds. Here, we used resting-state electroencephalographical (EEG) recordings from 144 healthy adults to test whether individual differences in intelligence (Raven’s Advanced Progressive Matrices scores) can be predicted from the complexity of temporally highly resolved intrinsic brain signals. We compared different operationalizations of brain signal complexity (multiscale entropy, Shannon entropy, Fuzzy entropy, and specific characteristics of microstates) regarding their relation to intelligence. The results indicate that associations between brain signal complexity measures and intelligence are of small effect sizes (r ∼ 0.20) and vary across different spatial and temporal scales. Specifically, higher intelligence scores were associated with lower complexity in local aspects of neural processing, and less activity in task-negative brain regions belonging to the default-mode network. Finally, we combined multiple measures of brain signal complexity to show that individual intelligence scores can be significantly predicted with a multimodal model within the sample (10-fold cross-validation) as well as in an independent sample (external replication, N = 57). In sum, our results highlight the temporal and spatial dependency of associations between intelligence and intrinsic brain dynamics, proposing multimodal approaches as promising means for future neuroscientific research on complex human traits.
Middle-aged persons with multimorbidity have to take their illnesses into account in their daily work, family and leisure activities. The MuMiA project aims to identify early preventive measures that make it easier for those between 30 and 60 years of age with multiple chronic diseases to manage their illnesses in their everyday lives. An interdisciplinary workshop and interviews with multimorbid middle-aged adults and their principal healthcare providers will be used to collect information on the management of care in the contexts of patients’ daily work, family and leisure activities. Data obtained in the interviews will be coded inductively and analysed using content analysis. Workshop outputs will be transcribed and evaluated by the authors. This study has received ethical approval from the Faculty of Medicine Ethics Committee of Goethe University (2021-47). The project will generate prevention recommendations that reflect the experiences of middle-aged persons living with multimorbidity and the views of their principal healthcare providers. The findings will be disseminated via conferences and peer-reviewed publications.
The mode of the antitumoral activity of multimutated oncolytic herpes simplex virus type 1 G207 has not been fully elucidated yet. Because the antitumoral activity of many drugs involves the inhibition of tumor blood vessel formation, we determined if G207 had an influence on angiogenesis. Monolayers of human umbilical vein endothelial cells and human dermal microvascular endothelial cells, but not human dermal fibroblasts, bronchial epithelial cells, and retinal glial cells, were highly sensitive to the replicative and cytotoxic effects of G207. Moreover, G207 infection caused the destruction of endothelial cell tubes in vitro. In the in vivo Matrigel plug assay in mice, G207 suppressed the formation of perfused vessels. Intratumoral treatment of established human rhabdomyosarcoma xenografts with G207 led to the destruction of tumor vessels and tumor regression. Ultrastructural investigations revealed the presence of viral particles in both tumor and endothelial cells of G207-treated xenografts, but not in adjacent normal tissues. These findings show that G207 may suppress tumor growth, in part, due to inhibition of angiogenesis.
Objectives: The aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days.
Materials and methods: Ex vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern.
Results: The membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane.
Conclusion: The present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs.
Clinical relevance: Understanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.
Objective: To investigate the value of standard [digital rectal examination (DRE), PSA] and advanced (mpMRI, prostate biopsy) clinical evaluation for prostate cancer (PCa) detection in contemporary patients with clinical bladder outlet obstruction (BOO) scheduled for Holmium laser enucleation of the prostate (HoLEP).
Material and Methods: We retrospectively analyzed 397 patients, who were referred to our tertiary care laser center for HoLEP due to BOO between 11/2017 and 07/2020. Of those, 83 (20.7%) underwent further advanced clinical PCa evaluation with mpMRI and/or prostate biopsy due to elevated PSA and/or lowered PSA ratio and/or suspicious DRE. Logistic regression and binary regression tree models were applied to identify PCa in BOO patients.
Results: An mpMRI was conducted in 56 (66%) of 83 patients and revealed PIRADS 4/5 lesions in 14 (25%) patients. Subsequently, a combined systematic randomized and MRI-fusion biopsy was performed in 19 (23%) patients and revealed in PCa detection in four patients (5%). A randomized prostate biopsy was performed in 31 (37%) patients and revealed in PCa detection in three patients (4%). All seven patients (9%) with PCa detection underwent radical prostatectomy with 29% exhibiting non-organ confined disease. Incidental PCa after HoLEP (n = 76) was found in nine patients (12%) with advanced clinical PCa evaluation preoperatively. In univariable logistic regression analyses, PSA, fPSA ratio, and PSA density failed to identify patients with PCa detection. Conversely, patients with a lower International Prostate Symptom Score (IPSS) and PIRADs 4/5 lesion in mpMRI were at higher risk for PCa detection. In multivariable adjusted analyses, PIRADS 4/5 lesions were confirmed as an independent risk factor (OR 9.91, p = 0.04), while IPSS did not reach significance (p = 0.052).
Conclusion: In advanced clinical PCa evaluation mpMRI should be considered in patients with elevated total PSA or low fPSA ratio scheduled for BOO treatment with HoLEP. Patients with low IPSS or PIRADS 4/5 lesions in mpMRI are at highest risk for PCa detection. In patients with a history of two or more sets of negative prostate biopsies, advanced clinical PCa evaluation might be omitted.