Preoperative radiosurgery for brain metastases (PREOP-1): A feasibility trial.

Purpose: Preoperative radiosurgery (SRS) of brain metastases (BM) aims to achieve cavity local control with a reduction in leptomeningeal relapse (LMD) and without additional radionecrosis compared to postoperative SRS. We present the final results of a prospective feasibility trial of linac-based stereotactic radiosurgery (SRS) prior to neurosurgical resection of a brain metastasis (PREOP-1). Methods: Eligibility criteria included a BM up to 4 cm in diameter for elective resection. The primary endpoint was the feasibility of delivering linac-based preoperative SRS in all patients prior to anticipated gross tumour resection. Secondary endpoints included rates of LMD, local control and overall survival. Exploratory endpoints were the level of expression of immunological and proliferative markers. Results: Thirteen patients of median age 65 years (range 41-77) were recruited. Twelve patients (92 %) received preoperative radiosurgery and metastasectomy and one patient went directly to surgery and received postoperative SRS, thus the primary endpoint was not met. The median time between referral and preoperative SRS was 6.5 working days (1-10) and from SRS to neurosurgery was 1 day (0-5). The median prescribed dose was 16 Gy (14-19) to a median planning target volume of 12.7 cm3 (5.9-26.1). Five patients completed 12-month follow-up after preoperative SRS without local recurrence or leptomeningeal disease. The patient who received postoperative FSRT developed LMD after six months. There was one transient toxicity (grade 2 alopecia) and nine patients have died from extracranial causes. Patients reported significant improvement in motor weakness at 6 months (P = 0.04). No pattern in changes of marker expression was observed. Conclusion: In patients with large brain metastasis without raised intracranial pressure, linac-based preoperative SRS was feasible in 12/13 patients and safe in 12/12 patients without any surgical delay or intracranial complications.

The development of a hiPSC-based platform to identify tissue-dependencies of IDH1 R132H.

The application of patient-derived (PD) in vitro tumor models represents the classical strategy for clinical translational oncology research. Using these cellular heterogeneous cultures for the isolation of cancer stem cells (CSCs), suggested to be the main driver for disease malignancy, relies on the use of surrogate biomarkers or is based on CSC-enriching culture conditions. However, the ability of those strategies to exclusively and efficiently enrich for CSC pool has been questioned. Here we present an alternative in vitro CSC model based on the oncogenic transformation of single clone-derived human induced pluripotent stem cells (hiPSC). Hotspot mutations in the DNA encoding for the R132 codon of the enzyme isocitrate dehydrogenase 1 (IDH1) and codon R175 of p53 are commonly occurring molecular features of different tumors and were selected for our transformation strategy. By choosing p53 mutant glial tumors as our model disease, we show that in vitro therapy discovery tests on IDH1-engineered synthetic CSCs (sCSCs) can identify kinases-targeting chemotherapeutics that preferentially target tumor cells expressing corresponding genetic alteration. In contrast, neural stem cells (NSCs) derived from the IDH1R132H overexpressing hiPSCs increase their resistance to the tested interventions indicating glial-to-neural tissue-dependent differences of IDH1R132H. Taken together, we provide proof for the potential of our sCSC technology as a potent addition to biomarker-driven drug development projects or studies on tumor therapy resistance. Moreover, follow-up projects such as comparing in vitro drug sensitivity profiles of hiPSC-derived tissue progenitors of different lineages, might help to understand a variety of tissue-related functions of IDH1 mutations.

Pelvic lymphadenectomy in vulvar cancer and its impact on prognosis and outcome.

BACKGROUND: The value of pelvic lymphadenectomy (LAE) has been subject of discussions since the 1980s. This is mainly due to the fact that the relation between lymph node involvement of the groin and pelvis is poorly understood and therewith the need for pelvic treatment in general. PATIENTS AND METHODS: N = 514 patients with primary vulvar squamous cell cancer (VSCC) FIGO stage ≥ IB were treated at the University Medical Center Hamburg-Eppendorf between 1996 and 2018. In this analysis, patients with pelvic LAE (n = 21) were analyzed with regard to prognosis and the relation of groin and pelvic lymph node involvement. RESULTS: The majority had T1b/T2 tumors (n = 15, 78.9%) with a median diameter of 40 mm (11-110 mm). 17/21 patients showed positive inguinal nodes. Pelvic nodal involvement without groin metastases was not observed. 6/17 node-positive patients with positive groin nodes also had pelvic nodal metastases (35.3%; median number of affected pelvic nodes 2.5 (1-8)). These 6 patients were highly node positive with median 4.5 (2-9) affected groin nodes. With regard to the metastatic spread between groins and pelvis, no contralateral spread was observed. Five recurrences were observed after a median follow-up of 33.5 months. No pelvic recurrences were observed in the pelvic nodal positive group. Patients with pelvic metastasis at first diagnosis had a median progression-free survival of only 9.9 months and overall-survival of 31.1 months. CONCLUSION: A relevant risk for pelvic nodal involvement only seems to be present in highly node-positive disease, therefore pelvic staging (and radiotherapy) is probably unnecessary in the majority of patients with node-positive VSCC.

Metachronous hepatic resection for liver only pancreatic metastases.

BACKGROUND: The value of liver resection (LR) for metachronous pancreatic ductal adenocarcinoma (PDAC) metastases remains controversial. However, in light of increasing safety of liver resections, surgery might be a valuable option for metastasized PDAC in selected patients. METHODS: We performed a retrospective, multicenter study including patients undergoing hepatectomy for metachronous PDAC liver metastases between 2004 and 2015 to analyze postoperative outcome and overall survival. All patients were operated with curative intent. Patients with oligometastatic metachronous liver metastasis with definitive chemotherapy (n = 8) served as controls. RESULTS: Overall 25 patients in seven centers were included in this study. The median age at the time of LR was 63.8 years (56.9-69.9) and the median number of metastases in the liver was 1 (IQR 1-2). There were eight non-anatomical resections (32%), 15 anatomical minor (60%) and 2 major LR (8%). Postoperative complications occurred in eleven patients (eight Clavien-Dindo grade I complications (32%) and three grade IIIa complications (12%), respectively). The 30-day mortality was 0%. The median length of stay was 8.6 days (IQR 5-11). Median overall survival following LR was 36.8 months compared to 9.2 months in patients with metachronous liver metastasis with chemotherapy (p = 0007). DISCUSSION: Liver resection for metachronous PDAC metastasis is safe and feasible in selected patients. To address general applicability and to find factors for patient selection, larger trials are urgently warranted.

Photoelectron spectroscopy of boron-containing reactive intermediates using synchrotron radiation: BH2, BH, and BF.

Mass selected slow photoelectron spectra (SPES) of three boron-containing reactive species, BH2, BH, and BF were recorded by double imaging photoion-photoelectron coincidence spectroscopy (i2PEPICO) using synchrotron radiation. All species were generated in a flow reactor from the H-abstraction of B2H6 by F atoms created in a F2 microwave discharge. The spectrum of BH2+ exhibits a long bending mode progression with a 970 cm-1 spacing due to the large geometry change from bent to linear upon ionization. Its ionization energy was determined as 8.12 ± 0.02 eV. For BH, photoionisation from both X1Σ+ singlet and a3Π triplet state was observed, permitting the experimental determination of the singlet/triplet gap (ΔEST) from the observed IE's of 9.82 eV and 8.48 eV. In addition, a threshold photoelectron spectrum of BF was recorded, which leads to an IE of 11.11 eV and an improved value for νBF+ of 1690 cm-1. All spectra were simulated by calculating Franck-Condon factors from optimised structures based on quantum chemical calculations.

Critical evaluation of an innovative mesh for bilateral transabdominal preperitoneal (TAPP) repair of inguinal hernias.

PURPOSE: Transabdominal preperitoneal hernia mesh plasty (TAPP) offers significant benefits to patients undergoing bilateral inguinal hernia repair. We evaluated a novel pre-shaped, large-pored, titanium-coated, lightweight polypropylene mesh for bilateral placement as an alternative to two separate meshes. METHODS: Thirty-six patients underwent elective surgical repair of bilateral inguinal hernias with the new mesh at three departments of surgery in Linz and Graz, Austria, between May 1, 2015 and June 30, 2017. RESULTS: All operations were completed without intraoperative complications or conversion to open procedures. The mean operation time was 74 min. There were no postoperative procedure-related complications with the exception of one hematoseroma of the spermatic cord. Two symptomatic medial recurrences (2/36 patients = 5.6%, 2/72 hernia repairs = 2.8%, respectively) after supravesical and medial hernia repair with the bilateral mesh were seen at structured follow-up examinations 6 and 12 months postoperatively. CONCLUSION: Treatment of bilateral inguinal hernias with the newly designed bilateral mesh for TAPP theoretically brings benefits in terms of resistance to forces acting on the mesh. The larger area may decrease the risk for mesh bulging and recurrence, and one large mesh might provide more stable support than two separate meshes overlapping at the midline. The results of our study do not confirm these theoretical benefits regarding a high recurrence rate (2.8%) after treatment of medial hernia defects. We recommend re-designing the mesh with only a small central slit, which would provide a broader mesh bridge with sufficient overlap for all types of inguinal and femoral hernias, including medial and supravesical defects. After the mesh has been re-designed, a new study should evaluate its real benefits before it is marketed.

Accelerated barrier recovery and enhancement of the barrier integrity and properties by topical application of a pH 4 vs. a pH 5·8 water-in-oil emulsion in aged skin.

BACKGROUND: Increased skin-surface pH is an important host-related factor for deteriorated barrier function in aged skin. OBJECTIVES: We investigated whether restoration of skin pH through topical application of a water-in-oil emulsion with pH 4 improved the barrier homeostasis in aged skin, and compared the effects with an identical galenic formulation with pH 5·8. METHODS: The effects of the test formulations on barrier recovery were investigated by repeated measurements of transepidermal water loss (TEWL) and skin pH 3 h, 6 h and 24 h after acetone-induced impairment of barrier function in aged skin. The long-term effects of the pH 4 and pH 5·8 emulsions were analysed by investigation of the barrier integrity and cohesion, the skin-surface pH and the skin roughness and scaliness before and after a 4-week, controlled application of the formulations. RESULTS: The application of the pH 4 emulsion accelerated barrier recovery in aged skin: 3 h and 6 h after acetone-induced barrier disruption the differences in the TEWL recovery between the pH 4 treated and acetone control fields were significant. Furthermore, long-term application of the pH 4 formulation resulted in significantly decreased skin pH, enhanced barrier integrity and reduced skin-surface roughness and scaliness. At the same time points, the pH 5·8 formulation exerted only minor effects on the barrier function parameters. CONCLUSIONS: Exogenous acidification through topical application of a water-in-oil emulsion with pH 4 leads to improvement of the skin barrier function and maintenance of the barrier homeostasis in aged skin.

Kinetics of the a-C3H5 + O2 reaction, investigated by photoionization using synchrotron radiation.

The kinetics of the combustion-relevant reaction of the allyl radical, a-C3H5, with molecular oxygen has been studied in a flow tube reactor at the vacuum ultraviolet (VUV) beamline of the Swiss Light Source storage ring, using the CRF-PEPICO (Combustion Reactions Followed by Photoelectron Photoion Coincidence Spectroscopy) setup. The ability to measure threshold photoelectron spectra enables a background-free detection of reactive species as well as an isomer-specific analysis of reaction products. Allyl was generated by direct photodissociation of allyl iodide at 266 nm and 213 nm and indirectly by the reaction of propene with Cl atoms, which were generated by photolysis from oxalyl chloride at 266 nm. Experiments were conducted at room temperature at low pressures between 0.8 and 3 mbar using Ar as the buffer gas and with excess O2 to maintain nearly pseudo-first-order reaction conditions. Whereas allyl was detected by photoionisation using synchrotron radiation, the main reaction product allyl peroxy was not observed due to dissociative ionisation of this weakly bound species. From the concentration-time profiles of the allyl signal, second-order rate constants between 1.35 × 1011 cm3 mol-1 s-1 at 0.8 mbar and 1.75 × 1011 cm3 mol-1 s-1 at 3 mbar were determined. The rates obtained for the different allyl radical generation schemes agree well with each other, but are about a factor of 2 higher than the ones reported previously using He as a buffer gas. The discrepancy is partly attributed to the higher collision efficiency of Ar causing a varying fall-off behavior. When allyl is produced by the reaction of propene with Cl atom, an unexpected product is observed at m/z = 68, which was identified as 1,3-butadienal in the threshold photoelectron spectrum. It is formed in a secondary reaction of allyl with the OCCl radical, which is generated in the 266 nm photolysis of oxalyl chloride.

Minispectrometer with handheld probe for 5-ALA based fluorescence-guided surgery of brain tumors: Preliminary study for clinical applications.

INTRODUCTION: Recently a mini-spectrometer with a handheld probe quantifying 5-aminolevulinic acid (5-ALA) based fluorescence intensity of brain tumors was developped by Kim et al. to improve fluorescence-guided neurosurgery. OBJECTIVE: To evaluate if this new tool is capable to discriminate nuances of fluorescence intensity of strongly fluorescing tumors (glioblastomas (GBM) and meningiomas (MM)). To study different modes of measurement (touch/no-touch). To determine protoporphyrin IX (PPIX) concentration in tumor tissue as compared to a laboratory spectrometer. MATERIAL AND METHODS: The tumor tissue was resected from patients operated in the neurosurgical department of University Hospital Duesseldorf, Germany between 01/2014 and 06/2014. Two spectrometers, one custom-built with a handheld probe ("mini-spectrometer") and one commercial laboratory spectrometer were employed. After calibration they were used to detect and compare fluorescence intensity of human brain tumor samples ex vivo under standardized conditions. The mini-spectrometer was tested at different distances to the tumor. PPIX concentrations of tumor lysates were determined by both spectrometers. RESULTS: In total n=11 tumors (5 MM and 6 GBM) resulting in 17 tumor biopsies were studied. All GBM showed significant higher fluorescence intensity as compared to MM (Z=-3.123, p=0.001). The fluorescence signal was inversely proportional to the square of the distance (GBM: R2=0.226; F=4.683; p<0.5; MM: R2=0255; F=8.042; p<0.01). The mini-spectrometer recorded fluorescence signals up to 2mm ("no-touch"). Determination of PPIX concentration in tumor by the mini-spectrometer did not differ from a laboratory spectrometer. CONCLUSION: The mini-spectrometer was a very sensitive tool for detection of 5-ALA based fluorescence of human brain tumors. Fluorescence intensity of glioblastoma and meningioma were significantly different. A no-touch mode of measurement was possible. PPIX concentration in tumor tissue could be determined as precisely as with a laboratory spectrometer. In future clinical trials the practicability of using such a tool in vivo has to be further evaluated.

Transplantation of neural progenitor cells in chronic spinal cord injury.

Previous studies demonstrated that neural progenitor cells (NPCs) transplanted into a subacute contusion injury improve motor, sensory, and bladder function. In this study we tested whether transplanted NPCs can also improve functional recovery after chronic spinal cord injury (SCI) alone or in combination with the reduction of glial scar and neurotrophic support. Adult rats received a T10 moderate contusion. Thirteen weeks after the injury they were divided into four groups and received either: 1. Medium (control), 2. NPC transplants, 3. NPC+lentivirus vector expressing chondroitinase, or 4. NPC+lentivirus vectors expressing chondroitinase and neurotrophic factors. During the 8 weeks post-transplantation the animals were tested for functional recovery and eventually analyzed by anatomical and immunohistochemical assays. The behavioral tests for motor and sensory function were performed before and after injury, and weekly after transplantation, with some animals also tested for bladder function at the end of the experiment. Transplant survival in the chronic injury model was variable and showed NPCs at the injury site in 60% of the animals in all transplantation groups. The NPC transplants comprised less than 40% of the injury site, without significant anatomical or histological differences among the groups. All groups also showed similar patterns of functional deficits and recovery in the 12 weeks after injury and in the 8 weeks after transplantation using the Basso, Beattie, and Bresnahan rating score, the grid test, and the Von Frey test for mechanical allodynia. A notable exception was group 4 (NPC together with chondroitinase and neurotrophins), which showed a significant improvement in bladder function. This study underscores the therapeutic challenges facing transplantation strategies in a chronic SCI in which even the inclusion of treatments designed to reduce scarring and increase neurotrophic support produce only modest functional improvements. Further studies will have to identify the combination of acute and chronic interventions that will augment the survival and efficacy of neural cell transplants.

17ß-Estradiol reduces nitric oxide production in the Guinea pig cochlea.

Intense noise exposure and the application of ototoxic substances result in increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO). In order to reduce the free NO concentration in the inner ear under pathological conditions, the use of natural cytoprotective substances such as 17ß-estradiol is a promising therapeutic concept. In male guinea pigs the organ of Corti and the lateral wall were isolated from the cochlea and afterwards incubated for 6 h in cell-culture medium. 17ß-Estradiol was adjusted in 2 concentrations to organ cultures of the right ears (12 animals per concentration). The left ears were used as controls. The NO production was quantified in the supernatant by chemiluminescence after incubation. Depending on the concentration, 17ß-estradiol reduced NO in the organ of Corti by 43% (p=0.015) and 46% (p=0.026), respectively. In the lateral wall, the NO concentration was reduced by 24%, but without statistical significance (p=0.86). However, when analyzing the association between the 2 cochlear regions for each animal separately, the NO concentrations were lower in nearly all 17ß-estradiol-treated ears compared to controls. In order to demonstrate the flexibility of the organ culture system, the NO donor DETA NONOate and the nitric oxide synthase inhibitors L-NAME and L-NMMA were applied. The electron microscopic analysis revealed a well-preserved cochlear cell morphology after incubation. The ability of 17ß-estradiol to influence the NO production preferentially in the organ of Corti might offer new therapeutic perspectives for inner ear protection.

Progresso temporal da ferrugem alaranjada em cultivares de cafeeiro no oeste de São Paulo / Temporal progress of orange rust in coffee cultivars in western Sao Paulo

A ferrugem é a doença mais comum das lavouras de café. O trabalho estudou nos cultivares de Coffea arabica L. Acaiá IAC 474-19, Catuaí Amarelo IAC 47, Icatu Amarelo IAC 2944, Icatu Vermelho IAC 4045, Obatã IAC 1669-20 e Apoatã IAC 2258 (Coffea canephora Pierre ex Fhroen) a incidência da ferrugem. O ensaio foi realizado em Adamantina, SP, onde os cafeeiros foram plantados no espaçamento de 4,0 x 2,0 m, com duas plantas por cova e dispostos em 6 linhas com 15 a 20 covas cada. No período entre junho de 2008 a dezembro de 2009, quinzenalmente, coletou-se ao acaso 30 folhas obtidas nos terços superior, médio e inferior dos cafeeiros e calculou-se a porcentagem das folhas com sintomas característicos da ferrugem. Calculou-se a área abaixo da curva de progresso da ferrugem para cada um dos cultivares. Constatou-se a doença na Icatu Amarelo IAC 2944 e Icatu Vermelho IAC 4045, cultivares descritos como resistentes/tolerantes à ferrugem. Concluiuse que a incidência diferiu entre os cultivares e diminuiu com o aumento da temperatura, mas não com a precipitação pluvial. A incidência da doença menor que 5% nos cafeeiros ocorreu com temperaturas máximas médias mensais maiores que 30º C

Rust is the most common disease in the coffee plantations. This work aimed to evaluate the incidence of coffee rust in the following cultivars of Coffea arabica L.: Acaiá IAC 474-19, Catuaí Amarelo IAC 47, Icatu Amarelo IAC 2944, Icatu Vermelho IAC 4045, Obatã IAC 1669-20 and Apoatã IAC 2258 (Coffea canephora Pierre ex Fhroen). The experiment was conducted in Adamantina, northwest region of the state of São Paulo, Brazil. The coffee trees were planted at a spacing of 4.0 x 2.0 m with two plants per hole. Each plot consisted of six lines with 15 to 20 holes each. From June 2008 to December 2009, 30 leaves were randomly collected from the upper, middle and bottom portions of the trees. The leaf rust was found in all cultivars, including on Catuai Amarelo IAC 2944 and Vermelho IAC 4045, although both cultivars are considered resistant to the disease in field conditions. The infections differed significantly among cultivars. The rust symptoms declined with increasing temperature, and the rainfall did not affect the incidence of disease. The less-than-5% infection rate coincided with the occurrence of maximum monthly mean temperatures greater than 30º C.

Reação de maracujazeiro amarelo a Meloidogyne incognita raça 3 / Reaction of yellow passion fruit to Meloidogyne incognita race 3

Neste trabalho estudou-se a reação de maracujazeiro amarelo 'Maguari' e 'Afruvec' ao fitonematoide Meloidogyne incognita raça 3, em casa-de-vegetação. Adotou-se o delineamento inteiramente casualizado, com três tratamentos (Maguari, Afruvec e Tomateiro cv. Rutgers) e quatro repetições, sendo a parcela constituída por um vaso contendo uma planta. Após seis meses, avaliou-se o índice de galhas e de massa de ovos nas cultivares de maracujazeiro amarelo e no tomateiro cv. Rutgers. A classificação da resistência ao fitonematoide foi feita utilizando o critério do fator de reprodução (FR). A 'Maguari' apresentou zero de índice de galhas e de massa de ovos, enquanto que 'Afruvec' mostrou baixo índice de galhas e massa de ovos comparativamente com o tomateiro cv. Rutgers. De acordo com o FR, a 'Maguari' enquadrou-se como imune ao nematoide, sendo a 'Afruvec' resistente e o tomateiro cv. Rutgers como suscetível.

This study concerned the reaction of yellow passion fruit 'Maguary' and 'Afruvec' to the phytonematode Meloidogyne incognita race 3 in greenhouse conditions. An entirely randomized experimental design with 3 treatments ('Maguary', 'Afruvec', and tomato cv. 'Rutgers') and 4 repetitions was used, each plot consisting of 1 vase containing 1 plant. After 6 months, an evaluation was made of the index of galls and egg mass in the yellow passion fruit varieties and in the tomato cv. 'Rutgers'. The classification of resistance to the phytonematode was made by criterion of the reproduction factor (RF). 'Maguary' presented a zero index of galls and egg mass, while 'Afruvec' showed a low index of galls and egg mass in relation to the tomato cv. Rutgers. According to the RF, 'Maguary' was characterized as immune to the phytonematode, while 'Afruvec' was resistant, and the tomato cv. 'Rutgers' was susceptible.

Comportamento do maracujazeiro amarelo, variedade Afruvec, ante uma população de Fusarium solani, agente causal da podridão do colo / Behaviour of yellow passion fruit, Afruvec variety, in relation to a population of Fusarium solani, causal agent of collar rot

O objetivo do presente trabalho foi verificar o comportamento do maracujazeiro amarelo, variedade Afruvec, ante uma população de Fusarium solani, obtida desse mesmo hospedeiro. O delineamento adotado foi o de blocos ao acaso, contendo dez tratamentos (nove isolados e tratamento testemunha), com quatro repetições, sendo cada parcela representada por um vaso contendo uma planta. Um disco do meio de cultura, colonizado com cada isolado do fungo, foi inoculado no colo ferido das plantas da variedade Afruvec, dois meses após a semeadura. Avaliou-se a patogenicidade, a incidência (número de plantas mortas) e a severidade da doença (comprimento da lesão no colo), até os sessenta dias após a inoculação. A variedade Afruvec foi suscetível ao fungo e apresentou variabilidade quanto à incidência e severidade da doença diante dos diferentes isolados. A população do fungo apresentou variabilidade em relação à agressividade. Com a evidência de diversidade genética na população do fungo, recomenda-se, também, nos testes de seleção de materiais ao patógeno, a avaliação desses materiais em diferentes localidades com histórico da doença ou inoculação com uma mistura de isolados do fungo, a fim de se conhecer a resistência ampla do genótipo ao patógeno.

The objective of the present work was to verify the behavior of yellow passion fruit, Afruvec variety, in relation to a population of Fusarium solani, obtained from this crop. The experimental delineation was random blocks, containing 10 treatments [9 isolates and 1 control treatment], with 4 repetitions, each plot being represented by a vase containing a plant. A disk of culture medium colonized by each isolate of the fungus was inoculated in the wounded collar region of the plants of the Afruvec variety two months after sowing. The appraised parameters were: the pathogenicity, the incidence (number of dead plants) and the severity of the disease (length of the lesion in the collar region), until 60 days after inoculation. The Afruvec variety was susceptible to the fungus and also presented variability as to the severity of the disease and incidence in relation to the different isolates. The population of the fungus showed variability in regard to aggressiveness. In light of the evidence of genetic diversity in the F. solani population, it is also suggested, in the tests of selection of materials to the pathogen, that these materials should be evaluated in different places with a history of the disease or inoculation with a pool of isolates of the fungus, in order to know the wide resistance of the genotype to the pathogen.

Stimulation of syncytium formation in vitro in human trophoblast cells by galectin-1.

BACKGROUND: Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In trophoblast cells, gal-1 binds to cell surface glycoproteins (e.g., Mucin1). It has been demonstrated that gal-1 recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast from second trimester human placenta and choriocarcinoma cells BeWo, which reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype. So the aim of this study was to investigate the effect of gal-1 on syncytium formation in BeWo and human villous trophoblasts (HVT) cells. MATERIALS AND METHODS: The effect of gal-1 on syncytium formation was investigated with immunocytochemical and double immunofluorescence stainings, cell-labelling and Real-time RT-PCR. BeWo choriocarcinoma and HVT cells were incubated in vitro for 24 and 48 h in the absence (controls) and presence of gal-1 and forskolin and stained with antibodies against Ki67, beta-catenin, E-cadherin and syncytin. BeWo and HVT cells were incubated with 60 microg/ml gal-1 for 48 h (BeWo) or 96 h (HVT) and cell fusion was detected by fluorescent cell-labelling solution. Finally, BeWo cells were incubated for 1 h or 48 h in the absence and presence of 60 microg/ml gal-1 and Real-time RT-PCR was performed. RESULTS: We showed with immunocytochemical staining a downregulation of beta-catenin expression in the 24 h BeWo cell culture and with double immunofluorescence staining an inhibition of the beta-catenin and E-cadherin expression in the 48 h BeWo cell culture stimulated with gal-1 or forskolin. The inhibition of E-cadherin was demonstrated on mRNA level in the 1 h BeWo cell culture too. Increased cell fusion was also showed with DiO and DiI fluorescent cell-labelling solution in the 48 h BeWo cell culture. In addition, we demonstrated the downregulation of Ki67 protein expression in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. We also showed the upregulation of syncytin protein and mRNA expression after incubation of the 48 h BeWo cell culture with gal-1 or forskolin. Similar results were obtained with HVT cells: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as demonstrated with beta-catenin and E-cadherin double immunofluorescence staining. This increase was also shown by fluorescent cell-labelling with DiO and DiI in the 96 h HVT cell culture compared to untreated cells. CONCLUSION: Our data suggest that gal-1 stimulates the syncytium formation in choriocarcinoma cells BeWo and HVT cells in vitro and inhibits the expression of beta-catenin, E-cadherin and in addition Ki67 in BeWo cells. Therefore gal-1 may be a major trigger for the process of trophoblast cell fusion.

Inhibiton of RET and JAK2 signals and upregulation of VEGFR3 phosphorylation in vitro by galectin-1 in trophoblast tumor cells BeWo.

BACKGROUND: Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, binds to cell surface glycoproteins (Mucin-1) on trophoblast cells. Although it has been demonstrated that gal-1 induces cell differentiation processes in these cells, no information on its signal transduction processes is available so far. As tyrosine phosphorylation is a major mechanism that controls multiple biological processes including cell differentiation, survival and proliferation, the aim of this study was to examine which human receptor tyrosine kinases (RTKs) were phosphorylated in trophoblast cells by gal-1. MATERIALS AND METHODS: BeWo choriocarcinoma cells were incubated for 24h in the absence (controls) and presence of 60microg/ml galectin-1. With the RayBio Human RTK Phosphorylation Antibody Array 1, the relative levels of phosphorylation of different human RTKs could be detected simultaneously. The signal intensities were compared and quantified with the Quantity One Version 4.5.2 program. Gal-1-treated and non-treated cells were incubated with antibodies against REarranged during Transfection (RET) and phosphorylated RET(Y905). Staining reaction was performed with the avidin-biotinylated peroxidase complex (ABC) reagent. RESULTS: We demonstrated that gal-1 inhibited RET and Janus Kinase 2 (JAK2) signals and upregulated Vascular endothelial growth factor receptor 3 (VEGFR3) signal in BeWo cells. We also showed the downregulation of phosphorylation on RET phosphotyrosine residue 905 in BeWo cells with phosphorylation specific antibodies and immunocytochemistry. CONCLUSION: Out of a number of 71 different RTKs, the stimulation of BeWo cells with gal-1 showed a significant alteration of signal intensity in only 3 RTKs: JAK2, RET and VEGFR3. Our data suggest that phosphorylation of these RTKs could be involved in cell differentiation processes that could be responsible for the already known effect of gal-1 on BeWo cells, the inhibition of proliferation.

Ki67 index in intracranial ependymoma: a promising histopathological candidate biomarker.

AIMS: The Ki67 tumour cell proliferation index is an independent prognostic factor in ependymoma patients. Essential prerequisites for validation of the Ki67 index as a histopathological biomarker are the reproducibility of this factor and its prognostic influence by different observers (proof of objective clinical and analytical performance). To this end, the aim was to analyse systematically inter- and intraobserver agreement and reproducibility of the prognostic impact of the Ki67 index in intracranial ependymoma. METHODS AND RESULTS: The study cohort contained 78 cases of intracranial ependymoma. In all cases, the Ki67 index was assessed by four experienced observers (EOs) and by four inexperienced observers (IOs) using the manual hot-spot method. There was considerable agreement on Ki67 index assessment. There was higher observer agreement among EOs compared with IOs. For each observer, survival analysis showed significant association of low Ki67 index with favourable patient outcome. CONCLUSIONS: Our data show that the Ki67 index in intracranial ependymoma is a reproducible and robust prognostic factor and can be considered a promising histopathological candidate biomarker. Attainment of biomarker status requires further translational studies in the context of prospective therapeutic trials.

Choriocarcinoma with metastasis in a rabbit (Oryctolagus cuniculi).

A 3-year-old female rabbit (Oryctolagus cuniculi) presented with apathy and indisposition for 2-3 days. Palpation revealed a mass in the caudal abdomen, namely, in the wall of the uterus. Ovariohysterectomy was performed, and the tissues were submitted for histopathologic examination. The mass consisted of 3 different (trophoblastic, syncytiotrophoblastic, and cytotrophoblastic) neoplastic cell types originating from the uterus. Immunohistochemistry was positive for cytokeratin in all 3 neoplastic cell types, and the syncytiotrophoblasts were positive also for human chorionic gonadotropin. Together these features allow the diagnosis choriocarcinoma. This report documents the first case of a spontaneous choriocarcinoma in a rabbit.

Long-term fate of neural precursor cells following transplantation into developing and adult CNS.

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.

MR imaging of lineage-restricted neural precursors following transplantation into the adult spinal cord.

Neural precursor cell (NPC) transplantation is a promising strategy for treatment of CNS injuries and neurodegenerative disorders because of potential for cell replacement. An important element of future clinical applications is development of a non-invasive procedure to follow NPC fate. We show that neuronal-restricted precursors (NRPs) and glial-restricted precursors (GRPs), NPCs with lineage restrictions for neurons and glia, respectively, can be labeled in vitro with the superparamagnetic iron oxide contrast agent Feridex. Following engraftment into intact adult spinal cord, labeled cells robustly survived in white and gray matter and migrated selectively along white matter tracts up to 5 mm. Localization of cells was reliably established using ex vivo magnetic resonance imaging of spinal cords. Imaging coincided with histological detection of iron and the human alkaline phosphatase transgene in most grafting sites, including the stream of migrating cells. Following transplantation, magnetically labeled cells exhibited mature morphologies and differentiated into neurons, astrocytes, and oligodendrocytes, similar to grafts of unlabeled NRPs and GRPs. Interestingly, Feridex-labeled cells, but not unlabeled cells, induced influx of ED1-positive macrophages/microglia. Small numbers of these phagocytic cells took up iron from grafted cells, while the majority of Feridex label was found in transplanted cells. We conclude that Feridex labeling does not inhibit NPC differentiation and can be used to reliably localize NPCs by MRI following engraftment into adult CNS, with the possible exception of areas of rapidly proliferating cells. The present results are relevant for MR-guided clinical application of transplantation strategies in treatment of spinal cord injury and other CNS pathologies.