Macrophage migration inhibitory factor is differentially expressed in normal and choriocarcinoma trophoblast cells.

Trophoblast cells are specific for placenta, the organ necessary for development of the fetus. Trophoblast derived choriocarcinoma is a rare cancer, with high metastatic potential, invading surrounding tissues and distant organs. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in a wide range of biological processes, which is increased in almost all human cancers. Expression of MIF in normal and choriocarcinoma trophoblast cells is investigated here, using normal extravillous trophoblast derived cell line HTR-8/SVneo, and choriocarcinoma cell lines JAR and JEG3. Expression of MIF and its receptors CD74 and CXCR2 was investigated at mRNA level using qPCR. Expression of MIF protein was studied using immunofluorescence and western blot, under reducing and native conditions, in whole cell lysates, subcellular fractions and conditioned media. The expression of MIF mRNA was similar in all three cell lines, while CD74 mRNA was more expressed in choriocarcinoma cells (14-fold for JAR, 12-fold for JEG3, p<0.01). CXCR2 mRNA was higher in JEG3 cell line compared to HTR-8/SVneo cells (6-fold, p<0.01). While the cellular level of MIF was similar, the level of secreted MIF was lower in JAR cell conditioned media compared to media of both HTR-8/SVneo (2.8-fold, p<0.01) and JEG3 cells (4.1-fold, p<0.001). Cellular distribution of MIF was similar between the studied cell types. MIF was predominantly cytoplasmic, but also detected in membrane, nuclear soluble and nuclear chromatin fraction. MIF appeared in high molecular weight complexes of >150 kDa under native conditions. A band of 140-145 kDa was consistently present in JEG3 cell lysates, while it was absent or very weak in other cell types. These results show that MIF/CD74 axis is shifted in choriocarcinoma, as previously shown for other cancers, and further justifies research towards the most effective MIF targeting therapeutics.

Didehydroroflamycoin pentaene macrolide family from Streptomyces durmitorensis MS405(T) : production optimization and antimicrobial activity.

AIMS: The aim of this study was to improve production of pentaene 32,33-didehydroroflamycoin (DDHR) in Streptomyces durmitorensis MS405 strain to obtain quantities sufficient for in depth analysis of antimicrobial properties. METHODS AND RESULTS: Through classical medium optimization conditions for stable growth, DDHR production within 7 days of incubation was established. Yields of 215 mg l(-1) were achieved in shake flask experiments in complex medium with mannitol as the primary carbon source. DDHR had poor antibacterial activity with minimal inhibitory concentrations (MIC) of 400 µg ml(-1) for Staphylococcus aureus and Bacillus subtilis, while MIC of 70 µg ml(-1) was determined for Candida albicans. Using flow cytometry and fluorescent microscopy, it was demonstrated that DDHR induced membrane damage in C. albicans followed by cell death. Combination studies with known antifungal nystatin showed that DDHR is a promising agent for the development of novel antimycotic treatments potentially less toxic for human cells. CONCLUSIONS: Pentaene didehydroroflamycoin has no antibacterial activity but can be further developed for the application in antifungal therapy. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the stable and production in high yields of a novel pentaene family that acts on Candida cell membranes and can be used in combination with known antifungals. Polyenes are still antifungal antibiotics of choice, and therefore, isolation and production of new lead structures are highly significant.

Expression of tumor associated antigens CA 15-3 and CA 19-9 in trophoblast of the normal human placenta.

Mucin 1 (MUC1) is abundantly expressed by various organs, including human placenta and endometrium. Since glycan modifications of MUC1 are potentially relevant for physiological as well as pathological processes, this study was aimed at establishing an expression profile of two MUC1 glycoepitopes, CA 15-3 and CA 19-9, in trophoblast throughout pregnancy. Immunohistochemical analysis of normal placenta demonstrated that trophoblast cells express both mucin antigens throughout gestation with a distinct staining pattern. The staining of villous trophoblast was non-uniform for both antigens, and stronger for CA 15-3. Only a proportion of extravillous trophoblast of the cell column, in decidual stroma or lining blood vessels was also stained. Whether the studied MUC 1 glycoforms can be linked to trophoblast cells invasion remains to be established.

Differential diagnosis of liver diseases using serum biomarkers.

PURPOSE: Many human blood proteins are synthesised in the liver. Their serum levels may decrease or increase due to liver disorders and some of them serve as useful biomarkers. Determination of serum concentration of different biomarkers has important role in diagnosis of liver diseases and in monitoring the course of disease. In this work 3 serum markers associated with liver disorders were compared. The aim was to assess whether these biomarkers exhibit specific distribution pattern in different types of liver disease: liver neoplasia (primary hepatocellular carcinoma [HC] or metastatic liver disease [MLD] from colon cancer), viral hepatitis C (HCV) and the parasitic infection echinococcosis. METHODS: Serum concentrations of alpha-fetoprotein (AFP), ferritin and insulin-like growth factor I (IGF-I) were determined in patients with liver disease and compared between patient groups and with healthy persons. RESULTS: Serum AFP and ferritin levels exhibited similar pattern of change in patients with liver neoplasia or HCV, and concentrations of these 2 markers were significantly increased compared to the control group (p < 0.01 in each case). On the other hand, the concentration of IGF-I was significantly decreased in patients with liver neoplasia or echinococcosis compared to the control group (p < 0.05 for both). The concentration of IGF-I was significantly lower and the concentration of ferritin significantly higher in patients with HC than in patients with MLD from colorectal cancer (p < 0.01 for both). CONCLUSION: The results have shown that each hepatic pathology studied exhibited specific profile of the analysed set of biomarkers. Therefore, the simultaneous determination of the 3 mentioned biomarkers may help in differential diagnosis of liver diseases.

Human trophoblast requires galectin-3 for cell migration and invasion.

Invasive extravillous cytotrophoblast of the human placenta expresses galectins-1, -3, and -8 in vivo and in vitro. This study aimed to investigate the potential role of galectin-3 in cell migration and invasion, using recombinant human galectin-3 (rhgalectin-3), small molecule galectin inhibitor I47, and galectin-3 silencing. HTR-8/SVneo cell migration was stimulated by rhgalectin-3 and reduced by I47, which could be neutralised by rhgalectin-3. Inhibitor specificity and selectivity for the galectins expressed in extravillous trophoblast were validated in solid phase assays using recombinant galectin-1, -3, -8, confirming selectivity for galectin-3. HTR-8/SVneo cell migration and invasion, and invasion by isolated trophoblast cells in primary culture were significantly reduced in the presence of I47, which could be restored by rhgalectin-3. Upon HTR-8/SVneo cell treatment with galectin-3 siRNA both LGALS3 and galectin-3 protein were dramatically decreased. Silencing of galectin-3 induced significant reduction in cell migration and invasion, which was restored by rhgalectin-3. The influence on known mediators of cell invasion, MMP2 and -9, and integrins α1, α5, and ß1 was followed in silenced cells, showing lower levels of MMPs and a large reduction in integrin subunit ß1. These results show that galectin-3 acts as a pro-invasive autocrine/paracrine factor in trophoblast in vitro.

Carcinoembryonic antigen and related molecules in normal and transformed trophoblast.

Carcinoembryonic antigen (CEA, CD66e) and CEA-related cell adhesion molecules (CEACAMs) are important mediators in remodeling of diverse human tissues, and modulators of cell proliferation and differentiation. Expression by normal and transformed trophoblast of gestational trophoblastic diseases (GTDs), isolated cytotrophoblast and choriocarcinoma cell lines is presented here. Immunocyto/histochemistry of normal placenta (n=9), invasive mole (n=8), choriocarcinoma (n=7), a placental site trophoblastic tumor, cytotrophoblast in primary culture and JAr and JEG-3 cells was performed using polyclonal anti-CEA and specific monoclonal anti-CEA antibodies. Data were analyzed and scored using Mann-Whitney Test. CEA and CEA-related molecules were identified by Western blot and immunoaffinity chromatography in JAr and JEG-3 cells and extracts of 1st and 3rd trimester of pregnancy tissue and cytotrophoblast cell lysates. CEA is expressed throughout pregnancy, in first trimester predominantly in syncytiotrophoblast, but also in villous cytotrophoblast and extravillous trophoblast. Data presented here demonstrate that CEA is significantly increased in transformed trophoblast of GTDs (p<0.05). Both cytotrophoblast in primary culture and choriocarcinoma cell lines express CEA, with staining of granular deposits in JAr and cell membrane in JEG-3. The results suggest that CEA (CD66e) and other CEA-related protein(s) could be involved in trophoblast differentiation.

Galectin-8 is expressed by villous and extravillous trophoblast of the human placenta.

Galectins (gals) as multifunctional animal lectins are of great potential significance for establishment and function of the placenta, due to their capacity to modulate cellular functions including proliferation, adhesion, and invasion. Human trophoblast is known to express gal-1, gal-3 and gal-13 proteins, as well as mRNAs for gal-14, -16 and -17. This study was undertaken to establish cellular distribution of gal-8, not previously associated with trophoblast, since we have recently detected gal-8 RNA and protein in cytotrophoblast cell material. Results of immunolocalization showed that gal-8 was expressed by villous and extravillous cytotrophoblast.

Human carcinoma-associated and salivary mucins detected by anti-bovine submaxillary mucin antibodies.

Polyclonal rabbit anti-bovine submaxillary mucin antibodies, anti-BSM IgG, were analyzed by autoradiography and densitometry (after SDS electrophoresis and blotting), ELISA, and IRMA assays for reactivity against native antigen BSM, deglycosylated and desialylated BSM, and human salivary and carcinoma-associated mucins. Known human tumor marker CA19-9 antigen reacted with rabbit anti-BSM IgG under different conditions, either soluble or immobilized. As soluble antigen, in IRMA it reacted with anti-BSM antibodies used both as a catcher and a tracer, as well as in combination with monoclonal anti-CA19-9 antibodies as a catcher. MUC1 mucin is a carrier of this carbohydrate antigen, CA19-9, or sialyl-Lewis(a), as well as of CA15-3 antigen, a known breast tumor marker. Autoradiography and densitometry demonstrated binding of anti-BSM IgG to intact MUC1 in a sample of commercial standard preparation of CA19-9 antigen. The same method and analysis demonstrated binding of anti-BSM IgG to MUC1 and to smaller antigens of 85-120 kD in samples containing CA15-3 antigen: commercial standard preparation, human breast tissue, and human milk. In a sample of whole human saliva, reactions of both isoforms of MUC7 were detected by autoradiography, as bands of 85 and 115 kD, and densitogram analysis also demonstrated reaction with MUC5B. Chemical modifications performed as periodate oxidation and desialylation of the BSM demonstrated carbohydrate (i.e., sialic acid) epitope sensitivity for anti-BSM IgG. The results presented in this work indicate that polyclonal anti-BSM antibodies are specific for sialylated carbohydrate structure on mucins and could serve as a tool for investigation of human carcinoma-associated and salivary mucins.