Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation

Lung ultrasound predicts clinical course but not outcome in COVID-19 ICU patients: a retrospective single-center analysis

BACKGROUND Point-of-care lung ultrasound (LU) is an established tool in the first assessment of patients with coronavirus disease (COVID-19). Purpose of this study was to evaluate the value of lung ultrasound in COVID-19 intensive care unit (ICU) patients in predicting clinical course and outcome. METHODS We analyzed lung ultrasound score (LUS) of all COVID-19 patients admitted from March 2020 to December 2020 to the Internal Intensive Care Unit, Ludwig-Maximilians-University (LMU) of Munich. LU was performed according to a standardized protocol at ICU admission and in case of clinical deterioration with the need for intubation. A normal lung scores 0 points, the worst LUS has 24 points. Patients were stratified in a low (0-12 points) and a high (13-24 points) lung ultrasound score group. RESULTS The study included 42 patients, 69% of them male. The most common comorbidities were hypertension (81%) and obesity (57%). The values of pH (7.42 ± 0.09 vs 7.35 ± 0.1; p = 0.047) and paO2 (107 80-130 vs 80 66-93 mmHg; p = 0.034) were significantly reduced in patients of the high LUS group. Furthermore, the duration of ventilation (12.5 8.3-25 vs 36.5 9.8-70 days; p = 0.029) was significantly prolonged in this group. Patchy subpleural thickening (n = 38; 90.5{\%}) and subpleural consolidations (n = 23; 54.8{\%}) were present in most patients. Pleural effusion was rare (n = 4; 9.5{\%}). The median total LUS was 11.9 ± 3.9 points. In case of clinical deterioration with the need for intubation, LUS worsened significantly compared to baseline LU. Twelve patients died during the ICU stay (29{\%}). There was no difference in survival in both LUS groups (75{\%} vs 66.7{\%}, p = 0.559). CONCLUSIONS LU can be a useful monitoring tool to predict clinical course but not outcome of COVID-19 ICU patients and can early recognize possible deteriorations

Integrin-linked kinase controls retinal angiogenesis and is linked to wnt signaling and exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR

Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR

The Feasibility of Percutaneous Dilatational Tracheostomy in Immunosuppressed ICU Patients with or without Thrombocytopenia

Background. Percutaneous dilatational tracheostomy (PDT) has become the preferred method in several intensive care units (ICUs), but data on PDT performed in immunosuppressed and thrombocytopenic patients are scarce. This study aimed to analyze the feasibility of PDT in immunosuppressed and thrombocytopenic patients compared to conventional open surgical tracheostomy (OST). Methods. We retrospectively analyzed the charts of patients who underwent PDT or OST between May 2017 and November 2020. Our outcomes were stoma site infections and bleeding complications. Results. 63 patients underwent PDT, and 21 patients underwent OST. Distribution of gender ratio, age, SAPS II, time of ventilation before tracheostomy, and preexisting hematooncological diseases was comparable between the two groups. After allogeneic stem cell transplantation (alloSCT), patients were more likely to undergo PDT than OST (p=0.033). The PDT cohort suffered from mucositis more frequently (p=0.043). There were no significant differences in leucocyte or platelet count on the tracheostomy day. Patients with coagulation disorders and patients under immunosuppression were distributed equally among both groups. Stoma site infection was documented in five cases in PDT and eight cases in the OST group. Moderate infections were remarkably increased in the OST group. Smears were positive in six cases in the PDT group;none of these patients had local infection signs. In the OST group, smears were positive in four cases;all had signs of a stroma site infection. Postprocedural bleedings occurred in eight cases (9.5%) and were observed significantly more often in the OST group (p=0.001), leading to emergency surgery in one case of the OST group. Conclusion. PDT is a feasible and safe procedure in a predominantly immunosuppressed and thrombocytopenic patient cohort without an increased risk for stoma site infections or bleeding complications

Reconstitution of HHV-6B-specific T cells in patients after allo-HSCT.

<p>(A) HHV-6 DNA and HHV-6-specific CD8 T cells in patient 1. Two peripheral blood samples (day 57, day 68) were available for staining with three HLA-B*08:01/peptide multimers, DFK and QTR from HHV-6B and RAK from EBV BZLF1. (B) Dot plots for multimer stainings of patient 1. (C) Detection of HHV-6 DNA and of HHV-6-specific CD8 T cells in patient 2. Two peripheral blood samples (day 182, day 266) were stained with HLA-B*08:01/peptide multimers carrying HHV-6B peptides DFK, SPR, EGR, and RSK. (D) Detection of HHV-6 DNA and time of HLA/peptide multimer analysis in patient 3. Peripheral blood samples were available for multimer staining from an early and a late time point (day 56, day 1221). (E) Multimer staining for detection of HHV-6-specific T cells (DFK) and EBV-specific T cells in patient 3 at day 56. (F) Multimer staining for HHV-6-specific T cells (14 epitopes as indicated) and EBV-specific T cells (RAK) in patient 3 at day 1221. (G) Exemplary dot plots of multimer stainings of patient 3 at day 1221.</p

HHV-6B peptide-specific polyclonal T cell lines and CD8 T cell clones.

<p>(A) Peptide-specific T cells in PBMCs from four healthy HLA-B*08:01-positive donors (a-d) were detected in an IFN-γ ELISPOT assay. PBMCs were stimulated with pools of 146 octameric or 153 nonameric HHV-6B/HLA-B*08:01 candidate peptides, 29 Epstein-Barr virus peptides (positive control), or no peptides. Mean+SD of 3 replicates is shown. (B) T cell cultures from donor 1 were stimulated with the complete octamer or nonamer peptide pools for 42 or 56 days as indicated, and then tested in IFN-γ ELISA or ELISPOT assays for reactivity to non-overlapping peptide subpools. HLA-B*08:01-positive activated B cells (mini-LCLs) were used to present peptides. Mean+range of 2 replicates is shown. (C) CD8 T cell clones were screened for reactivity to HHV-6B peptide pools in IFN-γ ELISA, as shown in this example for 12 T cell clones. Autologous CD40-activated B cells were used to present peptides. (D) To identify each T cell clone's target within the peptide libraries, T cells were stimulated with "crossed" peptide subpools. One positive signal each for horizontal and vertical subpools identifies the target peptide, as shown here for one T cell clone, which turned out to be specific for the QTR peptide from U41.</p