Mechanisms of tumor immune escape in triple-negative breast cancers (TNBC) with and without mutated BRCA 1.

PURPOSE: Triple negative breast cancers (TNBC) are associated with an adverse outcome, although these tumors are sensitive to chemotherapy. In part, this phenomenon could be caused by tumor immune escape. The current study investigates immunogenicity of TNBC cells in vitro and the presence of immunosuppressive factors in the tumor microenvironment (pAKT and B7H1 expression, infiltration with regulatory T cells, [Tregs]). METHODS: Natural killer (NK)-cell induced lysis was evaluated in estrogen receptor (ER) positive MCF 7 breast cancers, in MDA-MB231 and MDA-MB468 and in HCC-1937 (BRCA 1 mutated) and HCC-1806 TNBC cells. Expression of pAKT, B7H1 and infiltration with Tregs were determined by immunohistochemistry in human specimens of benign and malignant breast disease. RESULTS: NK-cell induced lysis was significantly increased (p < 0.05) in four TNBC cell lines compared to ER + MCF 7 cells. Fibroadenomas and mastectomy samples were not infiltrated with Tregs. Infiltration with Tregs was 0.92 ± 0.21 in ER/PR + breast cancers and significantly higher in TNBC without (2.30 ± 0.34) and also significantly higher with mutation of BRCA 1 (2.10 ± 0.34). Expression of pAKT was absent in benign controls and 1.23 ± 0.36 in ER/PR + breast cancers, 1.78 ± 0.40 in TNBC without and 2.40 ± 0.30 with mutated BRCA 1. No significant differences of B7H1 expression occurred among the breast cancer subgroups. CONCLUSION: TNBC cell stimulate the NK-cell immune response significantly stronger than ER positive breast cancer cells. This could explain why infiltration with immunosuppressive Tregs is increased in human specimens of TNBC with and without mutated BRCA 1. Accordingly, immunomodulatory treatment strategies should be further explored in TNBC.

Household pesticide contamination from indoor pest control applications in urban low-income public housing dwellings: a community-based participatory research.

We designed this community-based participatory research (CBPR) project aiming to generate evidence-based research results to encourage residents living in urban low-income public housing dwellings engaging in a community-wide integrated pest management (IPM) program with the intention to improve their health and quality of life, as well as household conditions. We enrolled 20 families and their children in this study in which we utilized environmental exposure assessment (surface wipe and indoor air) tools to quantitatively assessing residential pesticide exposure in young children before the implementation of an IPM program. We analyzed those samples for 19 organophosphate (OP) and pyrethroid pesticides. The most commonly detected pesticides were pyrethroids, particularly permethrin and cypermethrin with average concentrations of 2.47 and 3.87 µg/m(2), respectively. In many dwellings, we detected OPs, which are no longer available on the market; however, their levels are significantly lower than those of pyrethroids. None of the 20 families was free from pesticide contamination in their households, and pesticides were commonly detected in living room and children's bedroom. The correlation among household hygienic conditions, the sighting of live pests/pest debris, and the degree of indoor pesticide contamination highlights the failure of conventional chemical-based applications for pest controls. The results from the current study, as well as other recent studies, conducted in low-income public housing, child care centers, and randomly selected homes in the U.S. should accentuate the need for alternative pest management programs that incorporate safer and more sustainable protocols for pest controls.

Natural resistance to ascorbic acid induced oxidative stress is mainly mediated by catalase activity in human cancer cells and catalase-silencing sensitizes to oxidative stress.

BACKGROUND: Ascorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress. METHODS: Effective concentration (EC(50)) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay. RESULTS: The tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC(50) > 20 mmol/L and fifty-five percent had an EC(50) < 20 mmol/L. With an EC(50) of 2.6-5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC(50): 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L). CONCLUSIONS: Fifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.

Human decidua and invasive trophoblasts are rich sources of nearly all human matrix metalloproteinases.

Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. As for all invasive cell types, the ability of CTB to infiltrate the uterine wall is facilitated by the activity of matrix metalloproteinases (MMPs), which is regulated by tissue inhibitors of MMPs (TIMPs). There is evidence for the expression of several MMPs and TIMPs in decidua. However, published data are limited. Therefore, to set a foundation for future research, we screened a panel of healthy human deciduas obtained during first, second and third trimester of pregnancy in addition to isolated decidual cell populations for the expression of all known human MMPs and TIMPs by RT-PCR, western blot and immunohistochemistry. In the decidual samples, we detected almost all MMPs and all four TIMPs at mRNA level. While the expression of proMMP-3 and active MMP-13 and -23 was down-regulated in the course of pregnancy, the pro forms of MMP-8, -19 and -23, active MMP-9, -10, -12, -15, -16, -26 and -28, and pro- and active MMP-14 increased towards the end of gestation. All MMPs and TIMPs were expressed in uterine natural killer cells, decidual fibroblasts and/or trophoblasts, with the exception of MMP-20 and -25. In summary, a remarkably broad spectrum of MMPs was expressed at the human feto-maternal interface, reflecting the highly invasive and remodelling effect on placenta formation. It can be speculated that expression of MMPs correlates with the invasive potential of CTBs together with a crucial role in activation of labour at term.

Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines. METHODS: In total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography. RESULTS: We demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10). CONCLUSIONS: Ishikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments.

Specific subsets of immune cells in human decidua differ between normal pregnancy and preeclampsia--a prospective observational study.

BACKGROUND: Changes in the balance of decidual leucocyte populations may lead to an unfavourable uterine microenvironment which may be associated with the development of preeclampsia (PE). In this study, we therefore investigated the leucocyte subpopulations in decidual tissues of 33 women with preeclampsia and 66 control patients. METHODS: Decidua was either obtained via curettage during cesarean section or dissected from the surface of the basal plate of the placenta after spontaneous delivery. We used FACS analysis to quantify decidual leukocytes (CD45), NK cells (CD56+/CD16+ and CD56++/CD16-), antigen presenting cells (HLA-DR, DC-Sign, CD14) and T/B cells (CD8, CD4, alpha-beta-T-cell receptor, gamma-delta-T-cell receptor, CD25, CD19). RESULTS: The number of decidual cytotoxic CD8+T-lymphocytes (P < 0.02), alpha-beta -T-cell receptor positive T cells (P < 0.03) and of CD56+/CD16+ NK cells (P < 0.03) was lower in decidua from women with PE than in decidua from control patients. CONCLUSION: The observed reduction of specific leucocyte subsets could create a microenvironment which is unfavourable for an appropriate placentation and could thereby be involved in the development of preeclamptic symptoms.

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

BACKGROUND: Matrix 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. METHODS: To 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. RESULTS: In 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. CONCLUSION: MMP-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.

Upregulation of chemokine and cytokine production during pregnancy.

BACKGROUND: Following implantation, endometrial stroma is transformed into decidual tissue via a complex remodeling process. In parallel with that process, a significant increase in immune cells can be detected. Several studies suggest that chemokines and cytokines orchestrate the transformation of decidual tissue and the infiltration of leukocytes. In this study, we therefore compared chemokine and cytokine expression in the first- and third-trimester nonpregnant endometrium and decidua. METHODS: Investigation of the expression patterns of cytokines, chemokines and growth factors in endometrial tissue (after routine hysterectomy) and human decidual tissue (7-8 weeks of gestation and 38-40 weeks of gestation, respectively) was performed by protein array analysis. RESULTS: This analysis revealed a significant increase in monocyte-attracting chemokines (granulocyte-macrophage colony-stimulating factor, growth-related oncogene, growth-related oncogene-alpha and monocyte chemoattractant protein-2) , granulocyte-attracting chemokines (epithelial neutrophil activating peptide and interleukin 8) and proinflammatory factors (interleukin-1alpha, leptin) in decidual compared to endometrial tissue. Furthermore, concentrations of angiogenic substances (vascular endothelial growth factor and thrombopoietin) significantly peaked in first-trimester deciduas. CONCLUSION: This study demonstrates increased expression of chemokines and cytokines in decidual tissue compared to nonpregnant endometrium.

The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation.

BACKGROUND: Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC) in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta) family including activin A (ActA) and inhibin A (InA) are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype. METHODS: To test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex) as controls. RESULTS: Both glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected. CONCLUSION: These findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs.

Expression of VEGFR-1 (Flt-1) in breast cancer is associated with VEGF expression and with node-negative tumour stage.

BACKGROUND: Vascular endothelial growth factor (VEGF) promotes breast cancer progression by inducing angiogenesis via VEGF receptors on endothelial cells but also signals directly through receptors such as VEGFR-1 (Flt-1) expressed on tumour cells. The impact of autocrine signalling loops on treatment with VEGF inhibitors is still unclear. MATERIALS AND METHODS: Six breast cancer cell lines were tested for expression of VEGFR-1 by RT-PCR and Western blot. To assess clinical significance, 93 breast cancer lesions were evaluated for expression of VEGF and VEGFR-1 by immunohistochemistry. RESULTS: VEGFR-1 mRNA was found in all 6 cell lines, while protein expression was found in 5 cell lines. VEGF was expressed in 60% and VEGFR-1 in 39% of breast cancer specimens. VEGFR-1 expression was associated with VEGF expression and with node-negative tumour stage. CONCLUSION: Our data suggest that analysis of VEGF/VEGFR-1 expression might be relevant in identifying patients with different response rates upon treatment with antiangiogenic agents.

Beta-hydroxybutyrate (3-OHB) can influence the energetic phenotype of breast cancer cells, but does not impact their proliferation and the response to chemotherapy or radiation.

BACKGROUND: Ketogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2-6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro. METHODS: Seven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed. RESULTS: 3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation. CONCLUSIONS: We found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro.

Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.

BACKGROUND: LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration, which was reported to be overexpressed in 8-12 % of human breast cancers and thought to be exclusively located in cytoplasm. METHODS: In the present work we analyzed the cellular and histological expression pattern of LASP-1 and its involvement in biological behavior of human breast cancer through correlation with standard clinicopathological parameters and expression of c-erbB2 (HER-2/neu), estrogen- (ER) and progesterone-receptors (PR). For this purpose immunohistochemical staining intensity and percentage of stained cells were semi-quantitatively rated to define a LASP-1 immunoreactive score (LASP-1-IRS). LASP-1-IRS was determined in 83 cases of invasive ductal breast carcinomas, 25 ductal carcinomas in situ (DCIS) and 18 fibroadenomas. Cellular LASP-1 distribution and expression pattern was visualized by immunofluorescence and confocal microscopy and assessed through separate Western blots of nuclear and cytosol preparations of BT-20, MCF-7, MDA-MB231, and ZR-75/1 breast cancer cells. RESULTS: Statistical analysis revealed that the resulting LASP-1-IRS was significantly higher in invasive carcinomas compared to fibroadenomas (p = 0.0176). Strong cytoplasmatic expression of LASP-1 was detected in 55.4 % of the invasive carcinomas, which correlated significantly with nuclear LASP-1-positivity (p = 0.0014), increased tumor size (p = 0.0159) and rate of nodal-positivity (p = 0.0066). However, levels of LASP-1 expression did not correlate with average age at time point of diagnosis, histological tumor grading, c-erbB2-, ER- or PR-expression. Increased nuclear localization and cytosolic expression of LASP-1 was found in breast cancer with higher tumor stage as well as in rapidly proliferating epidermal basal cells. Confocal microscopy and separate Western blots of cytosolic and nuclear preparations confirmed nuclear localization of LASP-1. CONCLUSION: The current data provide evidence that LASP-1 is not exclusively a cytosolic protein, but is also detectable within the nucleus. Increased expression of LASP-1 in vivo is present in breast carcinomas with higher tumor stage and therefore may be related with worse prognosis concerning patients' overall survival.

Immunohistochemistry in human placental tissue--pitfalls of antigen detection.

Because incongruous controversial staining results are a common phenomenon in the placenta, methodical investigations are important to prevent researchers from obtaining misleading results. While investigating dendritic cells (DC) at the human fetomaternal interface, we observed staining of endothelial cells (EC) in chorionic villi for CD83. Given the high specificity of this antigen for DC, this did not seem credible. Previous studies had revealed the same surprising staining pattern with human leukocyte antigen (HLA)-G antibodies. We therefore analyzed human placental EC staining more closely. Both CD83 and HLA-G antibodies were of the same mouse IgG2b isotype. We also observed EC staining with a panel of control antibodies of the IgG2b isotype. This suggests a high affinity of human placental capillaries for mouse IgG2b. Several commonly used techniques for blocking nonspecific binding of antibodies could not prevent this nonspecific EC staining. A new preincubation step with purified human IgG was introduced. This abolished any placental EC staining with CD83, HLA-G, and IgG2b isotype control antibodies, presumably by blocking Fc receptors, whereas specific staining patterns remained unchanged. Mouse antibody of the IgG2b isotype are bound nonspecifically by vascular endothelial cells in human placenta and this can be overcome by blocking with purified human IgG. This blocking procedure could also be appropriate for frozen tissues other than placenta in which Fc receptors are expressed.

Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against Langerin/CD207.

Dendritic cells are professional antigen-presenting cells that initiate primary immunity. Migration from sites of antigen uptake to lymphoid organs is crucial for the generation of immune responses. We investigated the migratory pathways specifically of epidermal Langerhans cells by tracing them from the epidermis to the draining lymph nodes. This was possible with a new monoclonal antibody, directed against murine Langerin/CD207, a type II lectin specific for Langerhans cells. In situ, resident, and activated Langerhans cells express Langerin in the epidermis and on their way through dermal lymphatic vessels. Both emigrated and trypsinization-derived Langerhans cells expressed high levels of Langerin intracellularly but reduced it upon prolonged culture periods. Sizeable numbers of Langerin+ cells were found in skin draining lymph nodes but not in mesenteric nodes. Langerin+ cells localized to the T cells areas and rarely to B cell zones. Numbers of Langerin-expressing cells increased after application of a contact sensitizer. In the steady state, Langerhans cells in the skin-draining nodes expressed maturation markers, such as 2A1 and costimulatory molecules CD86 and CD40. These molecules, CD86 and CD40, were further upregulated upon inflammatory stimuli such as contact sensitization. Thus, the novel anti-Langerin monoclonal antibody permits the unequivocal visualization of migratory Langerhans cells in the lymph nodes for the first time and thereby allows to dissect the relative immunogenic or tolerogenic contributions of Langerhans cells and other types of dendritic cells.

Indoleamine 2,3-dioxygenase (IDO) expression in invasive extravillous trophoblast supports role of the enzyme for materno-fetal tolerance.

It is still not understood how the fetus escapes from being attacked by the maternal immune system. Recent reports based on mouse and in vitro models have suggested that the enzyme indoleamine 2,3-dioxygenase (IDO) is important for materno-fetal tolerance. IDO activity in the human placenta is known to be high and might lead to inhibition of T-cell proliferation, thus preventing fetal tissue from rejection by the maternal immune system. In an attempt to elucidate the precise location of IDO at the feto-maternal junctional zone, we investigated human placental and decidual tissue of first and third trimester of pregnancy using an immunohistochemical approach. In placental tissues, only syncytiotrophoblast and endothelial cells showed moderate expression of IDO. This pattern was observed regardless of whether first or third trimester tissue was investigated. In early and term decidua, cells with the typical morphology of invasive extravillous trophoblast (EVT) were strongly positive for IDO. Blocking immunohistochemical experiments with cytokeratin and IDO antibodies identified invasive EVT as the location of predominant IDO expression. Since EVT are the fetal cells with the closest contact to the maternal immune system, our results suggest that it is EVT which protects the fetus from rejection by downregulating local maternal T-cell responses.

Expression of tumor markers on breast and ovarian cancer cell lines.

BACKGROUND: Breast and ovarian tumor cell lines with defined tumor-associated antigens could serve as allogeneic antigen-sources for the biological therapy of cancer. MATERIALS AND METHODS: Eight breast and 3 ovarian cancer cell lines were stained for the antigens in question by immunohistochemistry. Antigen-expression was graded in 5 steps by light microscopic examination. RESULTS: All cell lines with the exception of PA-1 expressed EMA. Only MCF-7 was CEA-positive. MUC-1 was found in all cell lines apart from MDA-MB and SK-OV3. The highest expression of Her-2/neu was found in ZR-75/1 and SK-OV3. A491D and PA1 cells were CD19-negative, all other lines were positive. Only A491D and BT-20 had p53 accumulation. PR was found in 5 cell lines and ER in 6 cell lines. CONCLUSION: The cell lines investigated express a different pattern of tumor markers. In consequence, a careful examination should be performed in order to find the "best fitting" cell line to a patient's tumor for a tumor-vaccine.

Quantification of the predominant immune cell populations in decidua throughout human pregnancy.

PROBLEM: To date, a multiplicity of factors contributing to the establishment and progression of a successful pregnancy have been postulated. There is emerging evidence that decidual leukocytes could be decisive factors during pregnancy. Despite numerous investigations on immune cells in human early pregnancy decidua, little is known about the physiological composition and proportion of the various immune cell populations during the different phases of pregnancy. In this study, we therefore analyzed the proportion of the dominant decidual leukocytes in human tissue samples derived from all phases of pregnancy. METHODS: Single cell suspensions were prepared from decidual samples from 205 patients at 6-40 weeks of gestation. Cell populations were analyzed by flow cytometry, and immune cell populations were quantified as percentage of decidual CD45(+) cells. RESULTS: There was generally no difference in immune cell counts comparing decidua of healthy gestations and those with systemic inflammation. Overall, the proportion of uNK cells continuously decreased, while the amount of monocytes, immature dendritic cells, and T cells increased until term. Striking modifications in cell counts were seen during the 7th week compared with the 6th and later weeks of gestation. CONCLUSION: Studying the proportion of decidual immune cells during pregnancy, we detected a unique pattern which could be useful to design novel therapies for pathological conditions during pregnancy.

The expression of galectin-1 in vulvar neoplasia.

Vulvar cancer contributes to about 5% of all gynaecological cancers. Galectin-1, a member of an ubiquitous expressed beta-galactoside-binding family that comprises over 140 members to date, has been shown to be involved in many physiological and pathological processes, such as tumour progression, by promoting cancer cell invasion and metastasis, in apoptosis, embryogenesis and immunobiology. As the result of these findings, galectin-1 has been described as a potential marker for tumour progression in some malignancies. In this study, the expression pattern of galectin-1 was determined in 73 formalin-fixed, paraffin-embedded vulvar tissues by a standard immunohistochemical method: 12 benign vulvar specimen, 41 vulvar intraepithelial lesions (VIN), according to their differentiation were subdivided into VIN I, II and III and 20 invasive squamous cell carcinomas (ISCC). The immunohistochemical analyses showed that the intensity of galectin-1 expression on stromal cells next to the neoplastic cells steadily increased according to the pathological grade: benign vulvar tissue

Glycolytic phenotype in breast cancer: activation of Akt, up-regulation of GLUT1, TKTL1 and down-regulation of M2PK.

PURPOSE: Metabolic dependence on glucose utilisation has been described for different tumours characterised by activation of Akt, upregulation of GLUT1, M2PK and TKTL1. To date, however, little is known about glucose metabolism in breast cancer tissue. METHODS: We analysed 55 breast cancer specimens, 26 adjacent ductal carcinomas in situ (DCIS) and 23 adjacent normal breast tissues for expression of glycolytic markers by immunohistochemistry. RESULTS: We found expression of pAkt in 49%, GLUT1 in 25%, M2PK in 68% and TKTL1 in 31% of the tumours investigated. Expression of pAkt and Her2neu are positively correlated with borderline significance (P = 0.055). Expression of pAkt, GLUT1 and TKTL1 were higher in breast cancer and DCIS than in normal tissue. Surprisingly, M2PK expression was highest in normal breast tissue. CONCLUSIONS: We found a glycolytic phenotype in a high percentage of breast cancer samples. Inhibition of glycolysis might evolve as a future option for breast cancer therapy.

Antigen-presenting cells in pregnant and non-pregnant human myometrium.

PROBLEM: Inflammatory cells play a crucial role in human parturition. Different populations of leucocytes invade the reproductive tract. Numerous studies have described the decidual immune cell population in pregnant and non-pregnant endometrium. However, little is known about the presence of immune cells in human myometrium. METHOD OF STUDY: we herein analysed a spectrum of immune cells in human myometrium comparing tissue samples from non-pregnant (n = 8) and pregnant (n = 10) uteri. Applying immunohistochemistry with a panel of antibodies specific for T cells, monocytes, natural killer cells, B cells and antigen-presenting cells (CD4, CD8, CD14, CD15, CD16, CD19, CD56, CD68, CD83, HLA-DR, DC-Sign, mast cell tryptase), we characterized the immune cell population of human myometrium. RESULTS: a significantly higher number of CD14, CD15, CD16, DC-SIGN as well as CD4-positive cells were found in myometrium of pregnant compared to non-pregnant uteri, while mast cells were significantly reduced in pregnant myometrium. CONCLUSION: all markers found increased in pregnant myometrium indicate monocyte/macrophage lineage cells and thus suggest a possible involvement of these cells in healthy pregnancy maintenance. Monocytes/macrophages might produce a microenvironment that permits a controlled invasion of trophoblast cells into the myometrium while preventing a rejection of the semiallogenic conceptus and providing an important barrier against invading pathogenes.