Accidental Morcellation of Uterine Leiomyosarcoma Influences Relapse Free Survival but Does Not Negatively Influence Overall Survival.

Background: Uterine leiomyosarcoma (LMS) is a rare entity amongst malignant gynaecological tumours and is mostly diagnosed after surgery for benign leiomyoma (LM) of the uterus. As minimal invasive surgery is widely used, the morcellation of LM and the uterus is rather common. As there is little known about the impact of the morcellation of LMS on local and distant metastases, as well as overall survival, we carried out a large-scale retrospective study. Methods: A total of 301 LMS cases from the German Clinical Competence Centre for Genital Sarcomas and Mixed Tumours were analysed. We distinguished morcellated and non-morcellated LMS from pT1 and >pT1 tumours. Fine−Gray competing risks regressions and cumulative incidence rates were computed for the time to local recurrence, distant metastases, and patient death. Results: The recurrence free interval in pT1 LMS was significantly lower in the morcellation group with a 2-year cumulative incidence rate of 49% vs. 26% in non-morcellated LMS (p = 0.001). No differences were seen in >pT1 tumours. Distant metastases were more frequently found in non-morcellated pT1 LMS compared to the morcellated cases (5-year cumulative incidence: 54% vs. 29%, p < 0.001). There was no significant difference in time to death between both groups neither in the pT1 stages nor in >pT1 disease. Subdistribution hazard ratios estimated by multivariable competing risks regressions for the morcellation of pT1 LMS were 2.11 for local recurrence (95% CI 1.41−3.16, p < 0.001) and 0.52 for distant metastases (95% CI 0.32−0.84, p = 0.008). Conclusions: Tumour morcellation is not associated with OS for pT1 tumours. The morcellation of pT1 LMS seems to prolong the time to distant metastases whereas local recurrence is more likely to occur after the morcellation of pT1 LMS.

In vitro spheroid model of placental vasculogenesis: does it work?

Placental vascular development begins very early in pregnancy and is characterized by construction of a primitive vascular network in a low-oxygen environment. In vitro three-component assays of this process are scarce. In this study, a complex three-dimensional spheroid model for in vitro studies of placental vasculogenesis with regard to cell-cell interactions between cytotrophoblasts (CTs), villous stromal cells and endothelial precursor cells was established. Microscopic and immunohistochemical analyses of the spheroids showed structural and differentiation patterns resembling the structure and differentiation of early placental chorionic villous tissue (in regard to the expression of multiple markers cytokeratin-7, vimentin, CD34, CD31). The authenticity of this model to in vivo events allowed investigation of placental vascular development and trophoblast invasion under physiological and pathological conditions. Particularly enhanced spheroidal expression of SDF-1alpha and its receptor CXCR4, the major chemokine system in embryonic vasculogenesis, in a low-oxygen environment was detected. In addition, our model confirmed previously described invasive phenotype of trophoblasts through collagen under low- (physiologic), but not high- (pathologic) oxygen concentrations. Therefore, the three-dimensional spheroid model consisting of major placental cell types proved to be an appropriate system to investigate early placental vessel development under both physiological and pathological conditions.

Determination of In Vitro Membrane Permeability by Analysis of Intracellular and Extracellular Fluorescein Signals in Renal Cells.

BACKGROUND/AIM: The structural integrity of the eukaryotic cytoplasmic membrane is of crucial importance for its cell biological function and thus for the survival of the cell. Physical and chemical noxae can interact in various ways with components of the cytoplasmic membrane, influence its permeability and thus mediate toxic effects. In the study presented, changes in membrane permeability were quantified by intracellular accumulation of a fluorescent dye and by the release of the fluorescent dye from dye-loaded cells. MATERIALS AND METHODS: Non-malignant (RC-124) and malignant (786-O, Caki-1) renal cells were permeabilized with different concentrations of Triton X-100. The permeability of the membrane was determined at the single-cell level by the uptake of the dye into the cell inner by flow cytometry. In addition, a fluorescence plate reader was used to detect and quantify the release of the dye into the cell culture supernatant. RESULTS: Both malignant and non-malignant cells showed a dose-dependent alteration of membrane permeability after treatment with Triton X-100. In the presence of the fluorescent dye, significantly more dye was introduced into the permeabilized cells compared to control incubations. Vice versa, Triton X-100-treated and dye-loaded cells released significantly more dye into the cell culture supernatant. CONCLUSION: The combination of measurement of intracellular accumulated and extracellular released dye can quantifiably detect changes in membrane permeability due to cell-membrane damage. The combination of two different measurement methods offers additional value in reliable detection of membrane-damaging, potentially toxic influences.

Expression of transcription factor Oct-4 and other embryonic genes in CD133 positive cells from human umbilical cord blood.

A significant number of hematopoietic stem/progenitor cells (HSPC) in human umbilical cord blood could serve as a reservoir for the placental vasculature, yet, their morphological and functional features are not completely understood. Here, we describe the characterization of purified CD133(+) progenitor cells from umbilical cord blood, a subset of CD34(+) hematopoietic progenitors that were grown in proliferation medium containing Flt3-ligand, thrombopoietin and stem cell factor. Following isolation and enrichment of the CD133(+) cells by immunomagnetic cell sorting, they remained non-adherent for up to 40 days in culture and expressed different pluripotency markers including Sox-1, Sox-2, FGF-4, Rex-1 and Oct-4.Oct-4 expression was confirmed by laser-assisted single cell picking with subsequent quantitative real-time RT-PCR. The expression of Oct-4 indicates a pluripotent phenotype of CD133(+) cells and appears to be of functional relevance: After three weeks in endothelial differentiation medium, suspended cells became adherent, developed an endothelial cell-like morphology, bound fluoresceine isothiocyanate-labeled Ulex europaeus agglutinin-1, took up acetylated Di-LDL, and expressed other endothelial markers such as PECAM-1 or VEGFR-2. Concomitantly, Oct-4 expression was significantly reduced. Moreover, following treatment with retinoic acid, CD133(+) cells exhibited neural morphology associated with the expression of beta-III-tubulin. CD133(+) cells were found to express the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, detected by RT-PCR and immunocytochemistry. The recombinant human chorionic gonadotropin induced proliferation of the CD133(+) cells in a dose-specific manner. Our results indicate that CD133(+) HSPC from umbilical cord blood may have a greater differentiation potential than previously recognized and give rise to proliferative endothelial cells participating in placental vasculogenesis.