Impact of siRNA-Mediated Cofilin-1 Knockdown and Obesity Associated Microenvironment on the Motility of Natural Killer Cells.

The anti-tumor capacity of natural killer (NK) cells heavily relies on their ability to migrate towards their target cells. This process is based on dynamic actinrearrangement, so-called actin treadmilling, andis tightly regulated by proteins such as cofilin-1. The aim of the present study was to identify the role of cofilin-1 (CFL-1) in the migratory behavior of NK cells and to investigate a possible impact of an obesity-associated micromilieu on these cells, as it is known that obesity correlates with various impaired NK cell functions. CFL-1 was knocked-down via transfection of NK-92 cells with respective siRNAs. Obesity associated micromilieu was mimicked by incubation of NK-92 cells with adipocyte-conditioned medium from human preadipocyte SGBS cells or leptin. Effects on CFL-1 levels, the degree of phosphorylation to the inactive pCFL-1 as well as NK-92 cell motility were analyzed. Surprisingly, siRNA-mediated CFL-1 knockdown led to a significant increase of migration, as determined by enhanced velocity and accumulated distance of migration. No effect on CFL-1 nor pCFL-1 expression levels, proportion of phosphorylation and cell migratory behavior could be demonstrated under the influence of an obesity-associated microenvironment. In conclusion, the results indicate a significant effect of a CFL-1 knockdown on NK cell motility.

Challenges of Using Expansion Microscopy for Super-resolved Imaging of Cellular Organelles.

Expansion microscopy (ExM) has been successfully used to improve the spatial resolution when imaging tissues by optical microscopy. In ExM, proteins of a fixed sample are crosslinked to a swellable acrylamide gel, which expands when incubated in water. Therefore, ExM allows enlarged subcellular structures to be resolved that would otherwise be hidden to standard confocal microscopy. Herein, we aim to validate ExM for the study of peroxisomes, mitochondria, nuclei and the plasma membrane. Upon comparison of the expansion factors of these cellular compartments in HEK293 cells within the same gel, we found significant differences, of a factor of above 2, in expansion factors. For peroxisomes, the expansion factor differed even between peroxisomal membrane and matrix marker; this underlines the need for a thorough validation of expansion factors of this powerful technique. We further give an overview of possible quantification methods for the determination of expansion factors of intracellular organelles, and we highlight some potentials and challenges.

Selective retina therapy (SRT) in patients with therapy refractory persistent acute central serous chorioretinopathy (CSC): 3 months functional and morphological results.

PURPOSE: Central serous chorioretinopathy (CSC) is a disease presenting with detachment of the neurosensory retina and characteristic focal leakage on fluorescein angiography. The spontaneous remission rate is 84% within 6 months. In this study, the efficacy of selective retina therapy (SRT) was examined in patients with therapy refractory persistent acute CSC defined by symptoms for at least 6 months and persistent subretinal fluid (SRF) despite eplerenone therapy. MATERIAL AND METHODS: This is a prospective, monocentric observational study in 17 eyes (16 patients, mean age 42 years, 2 female). SRT was performed with the approved R:GEN laser (Lutronic, South Korea), a micropulsed 527-nm Nd:YLF laser device, with a train of 30 pulses of 1.7 µs at 100-Hz repetition rate at the point of focal leakage determined by fluorescein angiography (FA) at baseline (BSL). Visits on BSL, week 4 (wk4), and week 12 (wk12) included best corrected visual acuity (BCVA, logMar), central retinal thickness (CRT) on spectral domain optical coherence tomography (SD-OCT), and FA. Statistical analysis was performed by pair-by-pair comparisons of multiple observations in each case with Bonferroni correction for multiple testing. (IBM SPSS Statistics 25®). RESULTS: Mean CRT at BSL was 387.69 ± 110.4 µm. CRT significantly decreased by 106.31 µm in wk4 (95%-KI: 21.42-191.2; p = 0.01), by 133.63 µm in wk12 (95%-KI: 50.22-217.03; p = 0.001) and by 133.81 µm (95%-KI: 48.88-218.75; p = 0.001) compared to BSL. Treatment success defined as complete resolution of SRF occurred at wk4 in 7/17 eyes (35.3%) and at wk12 in 10/17 eyes (58.8%). Re-SRT was performed in 7/17 eyes (41.2%) after an average of 107.14 ± 96.59 days. Treatment success after Re-SRT was observed in 4/6 eyes (66.6%, 12 weeks after Re-SRT). Mean BCVA did not change significantly from BSL to any later timepoint after adjusting for multiple testing. Notably, eyes with treatment success showed better BCVA at all timepoints and gained more letters compared to failures. CONCLUSION: Single or repetitive SRT may be an effective and safe treatment in 2 of 3 patients suffering from acute persistent CSC after 6 months of symptoms or more. We observed complete resolution of SRF in around 60% of eyes 12 weeks after first SRT treatment and also 12 weeks after Re-SRT treatment in eyes with persistent or recurrent SRF. Results on the long-term course after SRT are still pending.

Hemopatch® as a Hemostatic Agent is Safe in Partial Nephrectomy: A Large, Single-Surgeon Retrospective Evaluation.

INTRODUCTION: Partial nephrectomy (PN) has evolved into the surgical standard of care for localized renal lesions. Hemostatic agents (HA) support the surgeon in achieving local hemostasis during PN. We previously reported initial results with the HA Hemopatch® in PN. We now report our experiences with Hemopatch® in a larger and more challenging single-surgeon PN cohort. METHODS: Our study included 45 patients who underwent PN due to suspicious renal lesions between December 2013 and March 2018. All surgeries were performed by a single surgeon using the HA Hemopatch®. Preoperative, intraoperative, and postoperative parameters were assessed. RESULTS: Preoperative median tumor diameter was 27 mm. Median PADUA and RENAL nephrometry scores were 7 and 6, respectively. In 13.3% of the cases an additional HA was applied. Intraoperative and postoperative bleeding occurred in 2.2% and 8.9%, respectively. Median total blood loss was 200 ml. Urgent pedicle clamping due to bleeding was necessary in 2 (4.4%) patients. The transfusion rate was 8.9%. There were no conversions. CONCLUSION: We confirmed our initial results demonstrating feasibility and reliability of Hemopatch® during PN. Notably, the cohort consists of selected patients. Prospective randomized studies are needed for comparison of different types of HA with regard to perioperative outcome.

Characterizing CD44 regulatory microRNAs as putative therapeutic agents in human melanoma.

The multistructural and multifunctional transmembrane glycoprotein CD44 is overexpressed in many tumors of distinct origin including malignant melanoma and contributes to a poor prognosis by affecting cell proliferation, cell migration, and also the sensitivity for apoptosis induction. Previous studies reported so far 15 CD44 regulatory microRNAs (miRs) in different cell systems. Using a novel method for miR affinity purification miR-143-3p was identified as most potent binder to the 3' untranslated region (UTR) of CD44. Overexpression of miR-143-3p in melanoma cells inhibits CD44 translation, which is accompanied by a reduced proliferation, migration and enhanced daunorubicin induced apoptosis of melanoma cells in vitro. Analyses of discordant CD44 and miR-143-3p expression levels in human melanocytic nevi and dermal melanoma samples demonstrated medium to high CD44 levels with no association to tumor grading or staging. The CD44 expression correlated to PD-L1, but not to MART-1 expression in malignant melanoma. Interestingly, the CD44 expression was inversely correlated to the infiltration of pro-inflammatory immune effector cells. In conclusion, the tumor suppressive miR-143-3p was identified as the most potent CD44 inhibitory miR, which affects growth characteristics of melanoma cells suggesting the implementation of miR-143-3p as as a potential anti-CD44 therapy of malignant melanoma.

A Missing Posterior Division of the Internal Iliac Artery.

INTRODUCTION: To date, multiple variations in the pelvic vasculature have been reported. In this case report, a rare, so far unreported, branching anomaly of the internal iliac artery on the left side of a male human pelvis is described. REPORT: The complete posterior division of the internal iliac artery was shown to be missing on dissection of a 73 year old male cadaver. The iliolumbar artery, the lateral sacral arteries, and the superior gluteal artery originated unilaterally (on the left pelvis) directly from the common iliac artery. DISCUSSION: No alteration was observed in the supplied structures of the left pelvic region, including the respective muscles, the sacrum, or the hip joint.

Leptin affects filopodia and cofilin in NK-92 cells in a dose- and time-dependent manner.

Hyperleptinemia, associated with obesity, is related with immune dysfunction and carcinogenesis. Natural Killer (NK) cells, a major component of the innate immune system are mediators of anti-tumor immunity and the most actively migrating cells among leukocytes. Actin rearrangement, promoted by cofilin plays a central role in cellular migration. Leptin affects the phosphorylation-dependent activity of cofilin and thus actin remodeling. We used human NK-92 cells to explore the in vitro effects of leptin on co-localization of cofilin and F-actin and on morphological changes in NK cells. NK-92 cells were incubated with different leptin concentrations (10 and 100 ng/mL) for 30 min and 24 h and immunocytochemically stained. Results demonstrate a dose- and time-dependent influence of leptin on cellular morphology. Utilizing confocal microscopy, we observed that the co-localization of cofilin-1 and F-actin was slightly influenced by leptin. In summary, the present study demonstrates an impact of a physiological leptin stimulation on the filopodia length, and a time-dependent effect on the co-localization of cofilin and F-actin in NK-92 cells.

Adiponectin and Its Receptors Are Differentially Expressed in Human Tissues and Cell Lines of Distinct Origin.

BACKGROUND: Adiponectin is secreted by adipose tissue and exerts high abundance and an anti-inflammatory potential. However, only little information exists about the expression profiles of adiponectin and its recently identified receptor CDH13 in non-tumorous human tissues and their association to clinical parameters. METHODS: The expression levels of adiponectin and CDH13 were analyzed in heart, liver, kidney, spleen, skin, blood vessels, peripheral nerve and bone marrow of 21 human body donors, in 12 human cell lines, and in purified immune effector cell populations of healthy blood donors by immunohistochemistry, Western-blot, and semi-quantitative PCR. The obtained results were then correlated to clinical parameters, including age, sex and known diseases like cardiovascular and renal diseases. RESULTS: Adiponectin expression in renal corpuscles was significantly higher in humans with known renal diseases. A coordinated expression of adiponectin and CDH13 was observed in the myocard. High levels of adiponectin could be detected in the bone marrow, in certain lymphoid tumor cell lines and in purified immune effector cell populations of healthy donors, in particular in cytotoxic T cells. CONCLUSION: For the first time, the expression profiles of adiponectin and CDH13 are analyzed in many human tissues in correlation to each other and to clinical parameters.