The first trimester human trophoblast cell line ACH-3P: a novel tool to study autocrine/paracrine regulatory loops of human trophoblast subpopulations--TNF-alpha stimulates MMP15 expression.

BACKGROUND: The trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1). RESULTS: Expression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e.g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts. CONCLUSION: We conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-alpha on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.

Approaching an individual methotrexate regimen in leptomeningeal carcinomatosis.

BACKGROUND: Chemotherapeutic effects in leptomeningeal carcinomatosis (LC) vary widely between patients, presumably in part because drug elimination from cerebrospinal fluid (CSF) differs between individuals. An individual dosing, adapted to elimination, may improve treatment efficacy. OBJECTIVE: To discuss the feasibility of easily accessible elimination parameters for an individual dosing of chemotherapy in LC. MATERIALS AND METHODS: The elimination of intrathecally applied methotrexate (Mtx) was tested in 14 LC patients and compared to the literature data. Plasma drug levels and CSF albumin levels are suggested as elimination parameters. RESULTS AND DISCUSSION: Mtx disappeared from CSF and appeared in plasma with an expected wide variation (interindividual range of coefficients of variation (CV) of CSF Mtx levels 158-189%, intraindividual range of CV of plasma Mtx levels 35-64%). Our data together with reported data suggest that plasma Mtx levels mirror closely the Mtx elimination from CSF. The levels of CSF albumin and of plasma Mtx at defined sample times correlated negatively (r=-0.7), which reflects their largely common elimination from CSF. CONCLUSION: Both parameters seem appropriate to describe the Mtx elimination from CSF. They should allow to individually adapt Mtx dosing towards an improvement of Mtx availability in CSF and of treatment efficacy.

Comparison of intravenous immunoglobulin preparations on microglial function in vitro: more potent immunomodulatory capacity of an IgM/IgA-enriched preparation.

Intravenous immunoglobulins (IVIg) have been used successfully as an immunomodulating treatment for patients with inflammatory diseases of the central nervous system (CNS) including multiple sclerosis (MS). It was shown previously that IVIg could modulate the functions of microglia, the main immune cell in the CNS. We have compared five commercially available IVIg preparations on their capacity to modulate tumor necrosis factor (TNF)-alpha secretion and nitric oxide production in cultured microglia. All preparations induced a dose-dependent stimulation of TNF-alpha secretion as measured by ELISA. There were some small differences between preparations consisting of IgG, while the preparation enriched for IgM and IgA induced a considerably higher TNF-alpha production at 1 mg/mL and 10 mg/mL. Similar results were seen for nitric oxide production as measured indirectly by the Griess reaction. These results indicate that IgM/IgA-enriched IVIg may be a more potent immunomodulator than pure IgG preparations on inflammatory reactions in the CNS.

Monoclonal antibody CD133-2 (AC141) against hematopoietic stem cell antigen CD133 shows crossreactivity with cytokeratin 18.

CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133-2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133-2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133-2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133-2/AC141 in IHC.

Extracellular pH modulates the secretion of fibronectin isoforms by human trophoblast.

Differentiation of human trophoblast from the proliferative to the invasive phenotype takes place in a hypoxic and thus likely an acidic microenvironment. During differentiation, the secretion pattern of fibronectin isoforms changes. Therefore, we analysed the relation between extracellular pH, secretion of fibronectin splice variants and invasiveness. By means of immunohistochemistry and biochemistry, cellular non-oncofetal fibronectins were found in placental stroma and around extravillous trophoblast, whereas oncofetal isoforms only marked the extracellular matrix of extravillous trophoblast. In vitro, mesenchymal cells produced non-oncofetal fibronectins only, whereas choriocarcinoma cell lines, extravillous trophoblast and choriocarcinoma/trophoblast hybrid cells secreted both non-oncofetal and oncofetal isoforms. When the pH of the culture medium was either lowered or increased (between 6.0 and 8.0), the trophoblast hybrids, but not choriocarcinoma and mesenchymal cells, responded with increased secretion of fibronectins and a shift towards oncofetal isoforms. These changes were preserved after pH normalisation. Histochemical determination of local tissue acidity revealed that the site of the lowest detectable tissue pK coincided with the starting point of invasion, the proximal part of trophoblastic cell columns. Therefore, it is concluded that the local pH plays an important role as regulator of differentiation of human trophoblast as reflected by the synthesis of oncofetal fibronectins by the invasive phenotype of extravillous trophoblast.