Johannes Dietl

Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature

BackgroundMatrix metalloproteinases (MMPs) are a family of structural and functional related endopeptidases. They play a crucial role in tumor invasion and building of metastatic formations because of their ability to degrade extracellular matrix proteins. Under physiological conditions their activity is precisely regulated in order to prevent tissue disruption. This physiological balance seems to be disrupted in cancer making tumor cells capable of invading the tissue. In breast cancer different expression levels of several MMPs have been found.MethodsTo fill the gap in our knowledge about MMP expression in breast cancer, we analyzed the expression of all known human MMPs in a panel of twenty-five tissue samples (five normal breast tissues, ten grade 2 (G2) and ten grade 3 (G3) breast cancer tissues). As we found different expression levels for several MMPs in normal breast and breast cancer tissue as well as depending on tumor grade, we additionally analyzed the expression of MMPs in four breast cancer cell lines (MCF-7, MDA-MB-468, BT 20, ZR 75/1) commonly used in research. The results could thus be used as model for further studies on human breast cancer. Expression analysis was performed on mRNA and protein level using semiquantitative RT-PCR, Western blot, immunohistochemistry and immunocytochemistry.ResultsIn summary, we identified several MMPs (MMP-1, -2, -8, -9, -10, -11, -12, -13, -15, -19, -23, -24, -27 and -28) with a stronger expression in breast cancer tissue compared to normal breast tissue. Of those, expression of MMP-8, -10, -12 and -27 is related to tumor grade since it is higher in analyzed G3 compared to G2 tissue samples. In contrast, MMP-7 and MMP-27 mRNA showed a weaker expression in tumor samples compared to healthy tissue. In addition, we demonstrated that the four breast cancer cell lines examined, are constitutively expressing a wide variety of MMPs. Of those, MDA-MB-468 showed the strongest mRNA and protein expression for most of the MMPs analyzed.ConclusionMMP-1, -2, -8, -9, -10, -11, -12, -13, -15, -19, -23, -24, -27 and -28 might thus be associated with breast cancer development and tumor progression. Therefore, these MMPs are proper candidates for further functional analysis of their role in breast cancer.

Unique appearance of proliferating antigen-presenting cells expressing DC-SIGN (CD209) in the decidua of early human pregnancy.

Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83(+) DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR(+)/DC-SIGN(+) APCs further phenotyped as CD14(+)/CD4(+)/CD68(+/-)/CD83(-)/CD25(-). These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209(+) cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN(+) decidual APCs were observed in situ to have intimate contact with CD56(+)/CD16(-)/ICAM-3(+) decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14(+)/DC-SIGN(+) decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14(+)/DC-SIGN(+) decidual cells into CD25(+)/CD83(+) mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset.

Human Decidua Contains Potent Immunostimulatory CD83+ Dendritic Cells

Dendritic cells (DCs) are sentinel cells of the immune system important in initiating antigen-specific T-cell responses to microbial and transplantation antigens. DCs are particularly found in surface tissues such as skin and mucosa, where the organism is threatened by infectious agents. The human decidua, despite its proposed immunosuppressive function, hosts a variety of immunocompetent CD45 cells such as natural killer cells, macrophages, and T cells. Here we describe the detection, isolation, and characterization of CD45(+), CD40(+), HLA-DR(++), and CD83(+) cells from human early pregnancy decidua with typical DC morphology. CD83(+) as well as CD1a(+) cells were found in close vicinity to endometrial glands, with preference to the basal layer of the decidua. In vitro, decidual CD83(+) cells could be enriched to approximately 30%, with the remainder of cells encompassing DC-bound CD3(+) T cells. Stimulation of allogeneic T cells in a mixed leukocyte reaction by the decidual cell fraction enriched for CD83(+) cells, was similar to that obtained with blood monocyte-derived DCs, demonstrating the potent immunostimulatory capacity of these cells. Decidual DCs with morphological, phenotypic, and functional characteristics of immunostimulatory DCs might be important mediators in the regulation of immunological balance between maternal and fetal tissue, leading to successful pregnancy.

Macrophage Migration Inhibitory Factor Contributes to the Immune Escape of Ovarian Cancer by Down-Regulating NKG2D

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) stimulates tumor cell proliferation, migration, and metastasis; promotes tumor angiogenesis; suppresses p53-mediated apoptosis; and inhibits antitumor immunity by largely unknown mechanisms. We here describe an overexpression of MIF in ovarian cancer that correlates with malignancy and the presence of ascites. Functionally, we find that MIF may contribute to the immune escape of ovarian carcinoma by transcriptionally down-regulating NKG2D in vitro and in vivo which impairs NK cell cytotoxicity toward tumor cells. Together with the additional tumorigenic properties of MIF, this finding provides a rationale for novel small-molecule inhibitors of MIF to be used for the treatment of MIF-secreting cancers.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A2A adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (Treg) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4+ and CD8+ T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated Treg. Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4+ T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of Treg but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

A New Rapid Immunohistochemical Staining Technique Using the EnVision Antibody Complex

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3′diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623–630, 2001)

Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now?

The target of any cryopreservation procedure should be to ensure high survival rates of living cells after thawing. Two important parameters determine the success of any cryopreservation protocol: the manner in which cells regain equilibrium in response to cooling, and the speed of freezing (cooling rate). Slow-rate freezing protocols result in the formation of ice crystals during cooling and warming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. However, there is a practical limit to the attainable cooling speed, and also a biological limit to the concentration of cryoprotectant tolerated by the cells during vitrification. Although post-warming survival depends on the species, the developmental stage and the quality of the embryos being vitrified, it seems clear that vitrification methods are increasingly successful and might be a better method than slow cooling procedures in the field of cryobiology. Many of the potential problems and benefits underlying vitrification as a method of choice for embryo cryopreservation in clinical embryology will be discussed in this review.

Leukaemia inhibitory factor (LIF) gene mutations in women with unexplained infertility and recurrent failure of implantation after IVF and embryo transfer.

OBJECTIVE Leukaemia inhibitory factor (LIF) plays a central role in the control of implantation. We undertook this study to investigate the prevalence of LIF gene alterations in women with unexplained infertility and with recurrent failure of implantation after in vitro fertilisation (IVF) and embryo transfer. PATIENTS AND METHODS Forty five women with recurrent failure of implantation after IVF (group A), 50 with unexplained infertility (group B) and 105 fertile women (controls) were screened for LIF gene mutations. Standard genomic DNA extraction, PCR amplification of the LIF gene and single-strand conformation polymorphism (SSCP) analysis were used to search for mutations which were subsequently confirmed by DNA sequencing. RESULTS In group A, one woman was identified as having a neutral LIF gene polymorphism in exon 3 without affecting protein conformation. In group B, one woman with a heterozygous mutation and one with a neutral polymorphism were detected. In controls, only one woman with a neutral polymorphism in the intron between exons 2 and 3 was found. The woman with a potentially functional LIF gene mutation in group B achieved an ongoing clinical pregnancy after ovarian superovulation. DISCUSSION Potentially functional mutations in the LIF gene do infrequently occur in women with unexplained infertility and may play a role in the etiology of infertility. However, routine screening for LIF mutations or polymorphisms in these women is not justified for the low prevalence of gene alterations.

Transketolase‐like 1 expression correlates with subtypes of ovarian cancer and the presence of distant metastases

Tumorbiology of ovarian cancer remains unclear. However, it is known that ovarian tumors, especially carcinomas, show elevated expression of glucose membrane transporters for facilitated glucose uptake. It can be assumed that increased glucose uptake leads to higher glucose metabolism. The energy resources of fully malignant transformed carcinomas are mainly supplied by aerobic glycolysis, for which several pathways are known. A key role in aerobic glycolysis is described for the transketolase enzymes. Recently, a novel transketolase-like enzyme called transketolase-like 1 (TKTL1) has been described that links aerobic glycolysis to the synthesis of fatty acids via production of acetyl-CoA. In order to investigate the role of TKTL1 for the progression of ovarian carcinomas, we examined paraffin sections of normal ovarian tissues, ovarian borderline tumors, and mucinous or serous papillary ovarian adenocarcinomas with respect to their expression of TKTL1. We identified a significantly elevated expression of TKTL1 in serous papillary ovarian adenocarcinomas, which correlates with poor prognostic parameters in the examined study group. Therefore, it can be assumed that TKTL1 plays a crucial role in ovarian cancer metabolism and that its expression predicts poor prognosis. Further investigations should be performed in order to evaluate whether this new enzyme is important for ovarian cancer tumorbiology and to analyze the potential role of TKTL1 as new target for specific antitumoral therapy

Maternal obesity and complications during pregnancy.

Abstract Obesity has become a major health problem all over the world and during pregnancy is associated with an increased risk of complications, including gestational diabetes, preeclampsia, and delivery complications such as macrosomia, shoulder dystocia and higher rates of cesarean sections and infections. Maternal obesity may also be an independent risk factor for neural tube defects and fetal mortality. This review focuses on the consequences of maternal obesity during pregnancy.