Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin.

Introduction: Hepatotoxicity induced by immunotherapeutics is an appearing cause for immune-mediated drug-induced liver injury. Such immuno-toxic mechanisms are difficult to assess using current preclinical models and the incidence is too low to detect in clinical trials. As hepatotoxicity is a frequent reason for post-authorisation drug withdrawal, there is an urgent need for immuno-inflammatory in vitro models to assess the hepatotoxic potential of immuno-modulatory drug candidates. We developed several immuno-inflammatory hepatotoxicity test systems based on recombinant human interleukin-2 (aldesleukin). Methods: Co-culture models of primary human CD8+ T cells or NK cells with the hepatocyte cell line HepaRG were established and validated with primary human hepatocytes (PHHs). Subsequently, the HepaRG model was refined by increasing complexity by inclusion of monocyte-derived macrophages (MdMs). The main readouts were cytotoxicity, inflammatory mediator release, surface marker expression and specific hepatocyte functions. Results: We identified CD8+ T cells as possible mediators of aldesleukin-mediated hepatotoxicity, with MdMs being implicated in increased aldesleukin-induced inflammatory effects. In co-cultures of CD8+ T cells with MdMs and HepaRG cells, cytotoxicity was induced at intermediate/high aldesleukin concentrations and perforin was upregulated. A pro-inflammatory milieu was created measured by interleukin-6 (IL-6), c-reactive protein (CRP), interferon gamma (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1) increase. NK cells responded to aldesleukin, however, only minor aldesleukin-induced cytotoxic effects were measured in co-cultures. Results obtained with HepaRG cells and with PHHs were comparable, especially regarding cytotoxicity, but high inter-donor variations limited meaningfulness of the PHH model. Discussion: The in vitro test systems developed contribute to the understanding of potential key mechanisms in aldesleukin-mediated hepatotoxicity. In addition, they may aid assessment of immune-mediated hepatotoxicity during the development of novel immunotherapeutics.

Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL.

Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.

GD2 Expression in Medulloblastoma and Neuroblastoma for Personalized Immunotherapy: A Matter of Subtype.

Neuroblastoma (NBL) and medulloblastoma (MB) are aggressive pediatric cancers which can benefit from therapies targeting gangliosides. Therefore, we compared the ganglioside profile of 9 MB and 14 NBL samples by thin layer chromatography and mass spectrometry. NBL had the highest expression of GD2 (median 0.54 nmol GD2/mg protein), and also expressed complex gangliosides. GD2-low samples expressed GD1a and were more differentiated. MB mainly expressed GD2 (median 0.032 nmol GD2/mg protein) or GM3. Four sonic hedgehog-activated (SHH) as well as one group 4 and one group 3 MBs were GD2-positive. Two group 3 MB samples were GD2-negative but GM3-positive. N-glycolyl neuraminic acid-containing GM3 was neither detected in NBL nor MB by mass spectrometry. Furthermore, a GD2-phenotype predicting two-gene signature (ST8SIA1 and B4GALNT1) was applied to RNA-Seq datasets, including 86 MBs and validated by qRT-PCR. The signature values were decreased in group 3 and wingless-activated (WNT) compared to SHH and group 4 MBs. These results suggest that while NBL is GD2-positive, only some MB patients can benefit from a GD2-directed therapy. The expression of genes involved in the ganglioside synthesis may allow the identification of GD2-positive MBs. Finally, the ganglioside profile may reflect the differentiation status in NBL and could help to define MB subtypes.

Reduction of adhesion formation after gynaecological adhesiolysis surgery with 4DryField PH - a retrospective, controlled study with second look laparoscopies.

Adhesions are a common consequence of abdomino-pelvic surgery. Efficacy of available adhesion prevention agents is discussed controversially. Here, we used the adhesion barrier 4DryField PH: a powder, which is transformed into a barrier gel with saline solution. The study includes 40 consecutive patients with surgeries for adhesiolysis, endometriosis and other gynaecological pathologies and subsequent second look interventions. The intervention group (n = 17) received 4DryField PH gel while control patients (n = 23) did not receive any adhesion prevention. Severity and extent of adhesion formation were scored during both interventions using an established score. Direct comparison between first and second interventions showed that extent and severity of adhesions could be reduced significantly using 4DryField PH gel. In contrast, in the control group, extent was not reduced and severity was even significantly higher. Direct comparison of second look laparoscopies revealed that adhesion extent and severity were significantly lower in the 4DryField PH than in the control group.Impact StatementWhat is already known on this subject? Adhesion formation after gynaecologic surgeries is known to be frequent and highly problematic as it directly induces complications and additionally makes subsequent surgeries more difficult. The effectiveness of established adhesion barriers is not sufficient to tackle these problems adequately.What the results of this study add? This is the first controlled study using the relatively new adhesion barrier 4DryField PH. It yields a significant reduction of extent as well as severity of adhesions, while adhesiolysis surgery alone does not solve the problem.What the implications are of these findings for clinical practice and/or further research? Usage of 4DryField PH gel seems to be a good approach to solve the adhesion problem of gynaecologic surgery in general and the reformation problem of adhesiolysis surgery specifically. The results should be confirmed in a larger prospective randomised controlled trial.

Changed inflammatory markers after application of 4DryField PH for adhesion prevention in gynecological surgery.

INTRODUCTION: The development of peritoneal adhesions and the effects of different antiadhesion agents on such mechanisms are not fully understood. Temporary rises of the C-reactive protein (CRP) level have been reported after antiadhesion agent application. We present the changes of inflammation markers observed after use of a starch-based polysaccharide certified for adhesion prevention and hemostasis 4DF (4DryField® PH). METHOD: Retrospective comparative analysis of inflammation markers in 40 patients undergoing laparoscopic adhesiolysis with or without adhesion prophylaxis was conducted. Statistical comparisons were performed by means of paired or unpaired t tests (for normally distributed continuous data), Wilcoxon matched pairs signed-rank tests or Mann-Whitney tests (for not-normally distributed continuous data), Mantel-Cox tests (for continuous data describing time intervals), and Fisher's exact tests (for discrete data). RESULTS: The maximum post-operative CRP level was significantly elevated in the 4DF group (87 vs. 29%; p < 0.001), whereas leukocyte concentration and body temperature did not differ between groups. No signs of infection were detected in any of the patients and CRP levels spontaneously dropped to normal values within few days. No side effects or complications were observed in both groups. In second-look surgeries performed for other diagnoses 1-56 weeks after the first interventions, no remnants of 4DF or any peritoneal inflammatory reactions were observed. CONCLUSION: The starch-based polysaccharide 4DF can be considered safe and does not induce inflammatory reactions of clinical significance. Further studies regarding 4DF degradation are recommended and, apart from macrophage migration, could also examine corresponding markers such as IL-6 and PCT.

Tumor Lipids of Pediatric Papillary Renal Cell Carcinoma Stimulate Unconventional T Cells.

Papillary renal cell carcinoma (PRCC) is a rare entity in children with no established therapy protocols for advanced diseases. Immunotherapy is emerging as an important therapeutic tool for childhood cancer. Tumor cells can be recognized and killed by conventional and unconventional T cells. Unconventional T cells are able to recognize lipid antigens presented via CD1 molecules independently from major histocompatibility complex, which offers new alternatives for cancer immunotherapies. The nature of those lipids is largely unknown and α-galactosylceramide is currently used as a synthetic model antigen. In this work, we analyzed infiltrating lymphocytes of two pediatric PRCCs using flow cytometry, immunohistochemistry and qRT-PCR. Moreover, we analyzed the CD1d expression within both tumors. Tumor lipids of PRCC samples and three normal kidney samples were fractionated and the recognition of tumor own lipid fractions by unconventional T cells was analyzed in an in vitro assay. We identified infiltrating lymphocytes including γδ T cells and iNKT cells, as well as CD1d expression in both samples. One lipid fraction, containing ceramides and monoacylglycerides amongst others, was able to induce the proliferation of iNKT cells isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and of one matched PRCC patient. Furthermore, CD1d tetramer stainings revealed that a subset of iNKT cells is able to bind lipids being present in fraction 2 via CD1d. We conclude that PRCCs are infiltrated by conventional and unconventional T cells and express CD1d. Moreover, certain lipids, present in pediatric PRCC, are able to stimulate unconventional T cells. Manipulating these lipids and T cells may open new strategies for therapy of pediatric PRCCs.

Liver-Specific Knockdown of Class IIa HDACs Has Limited Efficacy on Glucose Metabolism but Entails Severe Organ Side Effects in Mice.

Histone deacetylases (HDACs) are important regulators of epigenetic gene modification that are involved in the transcriptional control of metabolism. In particular class IIa HDACs have been shown to affect hepatic gluconeogenesis and previous approaches revealed that their inhibition reduces blood glucose in type 2 diabetic mice. In the present study, we aimed to evaluate the potential of class IIa HDAC inhibition as a therapeutic opportunity for the treatment +of metabolic diseases. For that, siRNAs selectively targeting HDAC4, 5 and 7 were selected and used to achieve a combinatorial knockdown of these three class IIa HDAC isoforms. Subsequently, the hepatocellular effects as well as the impact on glucose and lipid metabolism were analyzed in vitro and in vivo. The triple knockdown resulted in a statistically significant decrease of gluconeogenic gene expression in murine and human hepatocyte cell models. A similar HDAC-induced downregulation of hepatic gluconeogenesis genes could be achieved in mice using a liver-specific lipid nanoparticle siRNA formulation. However, the efficacy on whole body glucose metabolism assessed by pyruvate-tolerance tests were only limited and did not outweigh the safety findings observed by histopathological analysis in spleen and kidney. Mechanistically, Affymetrix gene expression studies provide evidence that class IIa HDACs directly target other key factors beyond the described forkhead box (FOXP) transcription regulators, such as hepatocyte nuclear factor 4 alpha (HNF4a). Downstream of these factors several additional pathways were regulated not merely including glucose and lipid metabolism and transport. In conclusion, the liver-directed combinatorial knockdown of HDAC4, 5 and 7 by therapeutic siRNAs affected multiple pathways in vitro, leading in vivo to the downregulation of genes involved in gluconeogenesis. However, the effects on gene expression level were not paralleled by a significant reduction of gluconeogenesis in mice. Combined knockdown of HDAC isoforms was associated with severe adverse effects in vivo, challenging this approach as a treatment option for chronic metabolic disorders like type 2 diabetes.

Activation of Adenosine Monophosphate-Activated Protein Kinase Reduces the Onset of Diet-Induced Hepatocellular Carcinoma in Mice.

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.

AMPKß1 and AMPKß2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, ß, and γ. AMPKα and ß are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKß1 and AMPKß2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKß2 was associated with a higher number of differentially regulated genes than the deletion of AMPKß1, suggesting that AMPKß2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKß2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKß1 impairs, whereas deficiency of AMPKß2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Helix 8 is the essential structural motif of mechanosensitive GPCRs.

G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H1 receptors (H1Rs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that H1Rs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)physiology.

Low level of exosomal long non-coding RNA HOTTIP is a prognostic biomarker in colorectal cancer.

Molecular risk stratification of colorectal cancer can improve patient outcome. A panel of lncRNAs (H19, HOTTIP, HULC and MALAT1) derived from serum exosomes of patients with non-metastatic CRC and healthy donors was analyzed. Exosomes from healthy donors carried significantly more H19, HULC and HOTTIP transcripts in comparison to CRC patients. Correlation analysis between lncRNAs and clinical data revealed a statistical significance between low levels of exosomal HOTTIP and poor overall survival. This was confirmed by multivariate analysis that HOTTIP is an independent prognostic marker for overall survival (HR: 4.5, CI: 1.69-11.98, p = 0.0027). Here, HOTTIP poses to be a valid biomarker for patients with a CRC to predict post-surgical survival time.

Expression of Glypican 3 is an Independent Prognostic Biomarker in Primary Gastro-Esophageal Adenocarcinoma and Corresponding Serum Exosomes.

Exosomes are nano-sized membranous vesicles of endosomal origin that carry nucleic acids, lipids and proteins. The cargo of exosomes is cell origin specific and the release of these exosomes and uptake by an acceptor cell is seen as a vital element of cell-cell communication. Here, we sought to investigate the diagnostic and prognostic value of the expression of glypican 3 (GPC3) on primary gastro-esophageal adenocarcinoma (GEA) tissue (tGPC3) and corresponding serum exosomes (eGPC3). Circulating exosomes were extracted from serum samples of 49 patients with GEA and 56 controls. Extracted exosomes were subjected to flow cytometry for the expression of eGPC3 and GPC3 expression on primary GEA tissue samples was determined by immunohistochemistry and correlated to clinicopathological parameters. We found decreased eGPC3 levels in GEA patients compared to healthy controls (p < 0.0001) and high tGPC3 expression. This was significantly associated with poor overall survival (high vs. low eGPC3: 87.40 vs. 60.93 months, p = 0.041, high vs. low tGPC3: 58.03 vs. 84.70 months, p = 0.044). Cox regressional analysis confirmed tGPC3 as an independent prognostic biomarker for GEA (p = 0.02) and tGPC3 expression was validated in two independent cohorts. Our findings demonstrate that eGPC3 and tGPC3 can be used as potential diagnostic and prognostic biomarkers for GEA.

Lack of beta-arrestin signaling in the absence of active G proteins.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

A first-in-human, randomized, controlled, subject- and reviewer-blinded multicenter study of Actamax™ Adhesion Barrier.

PURPOSE: Post-surgical adhesions remain a significant concern following abdominopelvic surgery. This study was to assess safety, manageability and explore preliminary efficacy of applying a degradable hydrogel adhesion barrier to areas of surgical trauma following gynecologic laparoscopic abdominopelvic surgery. METHODS: This first-in-human, prospective, randomized, multicenter, subject- and reviewer-blinded clinical study was conducted in 78 premenopausal women (18-46 years) wishing to maintain fertility and undergoing gynecologic laparoscopic abdominopelvic surgery with planned clinically indicated second-look laparoscopy (SLL) at 4-12 weeks. The first two patients of each surgeon received hydrogel, up to 30 mL sprayed over all sites of surgical trauma, and were assessed for safety and application only (n = 12). Subsequent subjects (n = 66) were randomized 1:1 to receive either hydrogel (Treatment, n = 35) or not (Control, n = 31); 63 completed the SLL. RESULTS: No adverse event was assessed as serious, or possibly device related. None was severe or fatal. Adverse events were reported for 17 treated subjects (17/47, 36.2%) and 13 Controls (13/31, 41.9%). For 95.7% of treated subjects, surgeons found the device "easy" or "very easy" to use; in 54.5%, some residual material was evident at SLL. For 63 randomized subjects who completed the SLL, adjusted between-group difference in the change from baseline adhesion score demonstrated a 41.4% reduction for Treatment compared with Controls (p = 0.017), with a 49.5% reduction (p = 0.008) among myomectomy subjects (n = 34). CONCLUSION: Spray application of a degradable hydrogel adhesion barrier during gynecologic laparoscopic abdominopelvic surgery was performed easily and safely, without evidence of clinically significant adverse outcomes. Data suggest the hydrogel was effective in reducing postoperative adhesion development, particularly following myomectomy.

Uterine perforation following a fractional curettage successfully treated with the modified polysaccharide 4DryField® PH: a case report.

BACKGROUND: Uterine perforation is the most common complication of curettage and may result in bleeding. Therefore, urgent control of bleeding from the uterine wall perforation is necessary to avoid an emergency hysterectomy or blood transfusion, to prevent peritoneal adhesion formation, possible chronic pelvic pain, and infertility. In the present case, an active bleeding secondary to a perforation of the uterus during curettage, for diagnosis of endometrial carcinoma, was instantaneously and successfully treated with only the application of a novel modified polysaccharide powder. This is, to the best of our knowledge, the first time that the agent 4DryField® has been used for this purpose. CASE PRESENTATION: A 71-year-old German woman with serometra and endometrial hyperplasia suffered a perforation of the anterior wall of the uterus during the hysteroscopic resection of submucosal polyps and a fractional curettage. Subsequently, an immediate laparoscopy showed an active bleeding from the wound, which was promptly stopped with only the application of the hemostatic and anti-adhesion polysaccharide powder, 4DryField®. There were no postoperative complications. Nine weeks later, a laparoscopic hysterectomy with bilateral salpingoophorectomy for endometrial carcinoma (histology: stage IA, pT1a, cN0, L0 V0 M0/G2) was performed. The former injured area looked slightly prominent, was completely healed, and showed a shiny serosa. All her pelvic organs were free of adhesions, and there was one 0.5-mm calcified granuloma in the Douglas pouch. CONCLUSIONS: The efficient hemostasis combined with the adhesion prevention effect of 4DryField®, allowed a fast control of the uterine wall bleeding, saved operation time, avoided the risks of other procedures for bleeding control and contributed to the normal healing of the uterine wall without any adhesion formation.

ß-Catenin Is Required for Endothelial Cyp1b1 Regulation Influencing Metabolic Barrier Function.

UNLABELLED: The canonical Wnt/ß-catenin signaling pathway is crucial for blood-brain barrier (BBB) formation in brain endothelial cells. Although glucose transporter 1, claudin-3, and plasmalemma vesicular-associated protein have been identified as Wnt/ß-catenin targets in brain endothelial cells, further downstream targets relevant to BBB formation and function are incompletely explored. By Affymetrix expression analysis, we show that the cytochrome P450 enzyme Cyp1b1 was significantly decreased in ß-catenin-deficient mouse endothelial cells, whereas its close homolog Cyp1a1 was upregulated in an aryl hydrocarbon receptor-dependent manner, hence indicating that ß-catenin is indispensable for Cyp1b1 but not for Cyp1a1 expression. Functionally, Cyp1b1 could generate retinoic acid from retinol leading to cell-autonomous induction of the barrier-related ATP-binding cassette transporter P-glycoprotein. Cyp1b1 could also generate 20-hydroxyeicosatetraenoic acid from arachidonic acid, decreasing endothelial barrier function in vitro In mice in vivo pharmacological inhibition of Cyp1b1 increased BBB permeability for small molecular tracers, and Cyp1b1 was downregulated in glioma vessels in which BBB function is lost. Hence, we propose Cyp1b1 as a target of ß-catenin indirectly influencing BBB properties via its metabolic activity, and as a potential target for modulating barrier function in endothelial cells. SIGNIFICANCE STATEMENT: Wnt/ß-catenin signaling is crucial for blood-brain barrier (BBB) development and maintenance; however, its role in regulating metabolic characteristics of endothelial cells is unclear. We provide evidence that ß-catenin influences endothelial metabolism by transcriptionally regulating the cytochrome P450 enzyme Cyp1b1 Furthermore, expression of its close homolog Cyp1a1 was inhibited by ß-catenin. Functionally, Cyp1b1 generated retinoic acid as well as 20-hydroxyeicosatetraenoic acid that regulated P-glycoprotein and junction proteins, respectively, thereby modulating BBB properties. Inhibition of Cyp1b1 in vivo increased BBB permeability being in line with its downregulation in glioma endothelia, potentially implicating Cyp1b1 in other brain pathologies. In conclusion, Wnt/ß-catenin signaling regulates endothelial metabolic barrier function through Cyp1b1 transcription.

Long term follow-up concerning safety and efficacy of novel adhesion prophylactic agent for laparoscopic myomectomy in the prospective randomized ADBEE study.

We conducted a prospective randomized single blind - subject study in the University Clinic of Gynecology of Pius-Hospital Oldenburg. The primary objective of the ADBEE study was to assess the safety and manageability of ADBLOCK when used as an adjunct to laparoscopic surgery for the primary of myomas in women wishing to improve pregnancy outcomes. The study population included 32 women aged between 18-45 years, in good general health condition, who have not completed their family planning and who are undergoing primary ('virgin') laparoscopic myomectomy with an aim to improve pregnancy outcomes. The patients were randomized in 2 groups, ADBLOCK arm with 21 patients and surgery only arm with 11 patients. The study was single blind - subject and the investigators were blinded to treatment group assignment until completion of uterine suturing and prior to removal of the endoscope. A vigorous follow-up of subjects was organized, focusing on its two critical characteristics: completeness and duration. Completeness represented the percentage of subjects who returned to every planed follow - up appointments. The patients were evaluated in a specific period of time, which defined the duration of follow-up. Safety of the ADBLOCK was estimated after analyzing and documentation of any adverse events occurred, clinical and physical examination of patients as well as evaluation of laboratory measures. There were 25 adverse events reported in ADBLOCK treatment group and 12 events in NO-ADBLOCK group over the 24-months treatment. All adverse events in both treatment arms were not anticipated, with all events in the ADBLOCK group being resolved. At 28 days, there was no significant difference in proportion of events between the two treatments (p = 0.440). Overall, the number of events reported was low and the severity of events was generally mild with an unlikely or no relationship to treatment. There were no unanticipated device related adverse events seen in both treatment groups over the immediate post-operative period or during the 24 months follow up period. By 12 weeks all patients reported their wound as healing well or healed and at 6 months all wounds were reported as healed. There were no differences between both treatment groups regarding the use of painkillers over 24 months follow up period. This clinical first - in - human study, sustained by a rigorous follow-up of the subjects has demonstrated that ADBLOCK is a safe product, presenting no additional safety risk or burden to the patients over surgery alone. The device was relatively easy to use, with a low device failure rate that had no impact on the surgical procedures.

Evaluation of the HystSim™-virtual reality trainer: an essential additional tool to train hysteroscopic skills outside the operation theater.

BACKGROUND: Minimally invasive surgery is a major pillar of gynecological surgery. However, there are very few training opportunities outside the operation theater (OR) due to the cost and equipment requirements of organ simulators, virtual reality trainers (VRT) are promising tools to fill this gap. METHODS: Experienced and inexperienced participants of a minimally invasive surgery course followed the standardized HystSim™-VRT training program. RESULTS: Performance of 39 Participants (15 inexperienced and 24 experienced) was evaluated in the standardized hysteroscopic program HystSim™. Tasks included three rounds of both a polyp and a myoma resection. Primary measurements were improvement in resection time, cumulative resection path length, and distention media use. CONCLUSION: The HystSim™-VRT is an effective tool to improve the psychomotor skills needed in hysteroscopic surgery for experienced and inexperienced surgeons prior to OR exposure. Additional organ models training is advisable for hysteroscopic haptic skills.

Use of Modified Polysaccharide 4DryField (®) PH for Adhesion Prevention and Hemostasis in Gynecological Surgery: A Two-Center Observational Study by Second-Look Laparoscopy.

Purpose. This study evaluates both scopes of 4DryField PH, certified for adhesion prevention and hemostasis, in patients undergoing surgery for various and severe gynecological disorders. Methods. This is a two-institutional study. Adhesion prevention efficacy was evaluated using video documentation of first-look laparoscopies (FLL) and second-look laparoscopies (SLL); other patient data were analyzed retrospectively. Twenty patients with various disorders were evaluated, 4 assigned to a uterus pathology, 10 to endometriosis, and 6 to an adhesion disease group. Nine patients received 4DryField primarily for hemostasis and 11 solely for adhesion prevention. Nineteen patients had SLL after 5 to 12 weeks and one after 13 months. Results. At FLL with 4DryField, immediate hemostasis could be achieved in diffuse bleeding. At SLL, effective adhesion prevention was observed in 18 of all 20 women, with only 2 revealing major adhesions. In particular, only 1 of the 6 women with adhesion disease as predominant disorder showed major adhesions at SLL. Conclusions. Modified polysaccharide 4DryField is not only effective in diffuse bleeding. In this cohort with extensive surgery for various gynecological pathologies, 4DryField showed effective adhesion prevention as confirmed at SLL, too. Its use as premixed gel is a convenient variant for treatment of large peritoneal wounds.