Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer.

AIMS: Epithelial cell adhesion molecule (EpCAM) is a widely used immunohistochemical marker for epithelial human malignancies. Antibodies to target EpCAM are usually directed against its ectodomain (EpEX), but do not detect the intracellular domain (EpICD). The aim of this study was to compare membranous EpEX versus EpICD expression by immunohistochemistry. METHODS AND RESULTS: Concurrent EpEX and EpICD expression was investigated retrospectively in cancerous and matched non-neoplastic tissue samples from patients with pancreatic adenocarcinoma. In total, 317 paired samples of pancreatic tissue from 88 patients were analysed and correlated with clinicopathological parameters. In non-cancerous tissue, a high concordance of membranous EpEX and EpICD expression was observed and defined as the expression of the full-length EpCAM (EpEX(+)/EpICD(+) phenotype, EpCAM(MF)), which was highly predominant. In contrast, while most tumour samples were EpEX positive, loss of membranous EpICD expression (EpEX(+)/EpICD(-) phenotype, EpCAM(MT)) was observed in one-third of cases, and these patients had a significantly shortened disease-free and overall survival. CONCLUSIONS: This study demonstrates for the first time that loss of membranous EpICD expression is a frequent event and predicts poor prognosis in patients with pancreatic cancer. Additional studies evaluating the predictive and prognostic value of the expression of different membranous EpCAM variants are warranted in epithelial cancers.

Characterization of antigen-presenting cells in fresh and cultured human corneas using novel dendritic cell markers.

PURPOSE: Adult healthy human corneas bear a distinctive number of antigen-presenting cells (APCs) important for the fate of a graft. The purpose of this study was to differentiate between Langerhans cells (LCs) and other dendritic cells (DCs) and between mature and immature APCs in fresh and cultured human corneas using specific markers. METHODS: Immunofluorescence double staining was performed for Langerin/CD207, CD1a, DC-SIGN/CD209, DC-LAMP/CD208, CD45, CD11c, CD11b and HLA-DR. RESULTS: Langerin(+)/CD1a(+)/HLA-DR(+) LCs (approximately 100 cells/mm(2) in fresh corneas) were found in the limbal and peripheral regions of corneal epithelium and the anterior stroma up to 83 days of culture. All these cells coexpressed CD45 and CD11c. DC-SIGN(+)/CD45(+) DCs (approximately 150 cells/mm(2) in fresh corneas) were detected mainly peripherally and in the anterior stroma, even in long-term cultured corneas. Most of these cells were HLA-DR(-). Few mature DCs (DC-LAMP(+)/HLA-DR(+)) were found in fresh and cultured corneas. Macrophages (CD11c(-)/CD11b(+)) were seen in the peripheral, paracentral, and even central regions of the posterior stroma. CONCLUSIONS: This is the first demonstration that human corneas harbor populations of Langerin(+)/CD1a(+)/HLA-DR(+) LCs and DC-SIGN(+) DCs in a distribution pattern similar to that in the skin. Few APCs are in a mature state (DC-LAMP(+)). Given the reduced but not complete depletion of APCs during organ culture, these grafts still bear a potential risk for rejection.

Development of a novel perfused rotary cell culture system.

A rotary cell culture system has been established. System quality was determined by observing the stability of the basic parameters of temperature, gas exchange, and pH, and mass transfer (time to equimolarity) between the medium circuit and the 2 cell-containing chambers was investigated. Mass transfer time for urea and several ions was approximately 30 min for the high-fiber-density chamber (HFC) and 50 min for the low-fiber-density chamber (LFC). Exchange of albumin was delayed in both chambers, highlighting the dependence of mass transfer on area of exchange and molecule size. Finally, the ability for cell growth and maintenance was tested. Densities of up to 1.2 x 10(7) immortalized cells per mL at a viability of up to 85% were obtained after 1 week of continuous, non-interfering culture of immortalized cells in the HFC. Human pancreatic islets were also cultivated in the LFC. Confocal analysis using fluorescent dyes showed that the 3-dimensional islet structure was maintained for 1 week. Promising results were obtained, which will further our ongoing efforts toward establishing a mobile cell culture system.

Maturation of dendritic cells in the presence of living, apoptotic and necrotic tumour cells derived from squamous cell carcinoma of head and neck.

Dendritic cells (DC) have been recently used as vaccines for stimulation of tumour-specific immunity in various types of cancer. Since data about interactions of DC with tumour cells derived from head and neck cancer are not available, in our study we investigated the effects of head and neck squamous cell carcinoma (HNSCC) cell lines on the maturation of DC. We found that immature DC efficiently internalise necrotic cells, but not living and apoptotic tumour cells. Although apoptotic cells induced a partial maturation of DC, they were not able to stimulate the secretion of IL-12. In contrast, necrotic tumour cell preparations from all three HNSCC cell lines induced the mature phenotype and IL-12 production by DC. Moreover, necrotic cells synergistically augmented stimulatory effects of monocyte-conditioned medium on the maturation of DC. Thus, DC-based vaccination utilizing necrotic tumour cells as a source of tumour antigens, even in combination with inflammatory stimulus, seems to be a suitable strategy for adjuvant immunotherapy in HNSCC.

Immunosuppressive effects of soluble factors secreted by head and neck squamous cell carcinoma on dendritic cells and T lymphocytes.

Recent observations suggest that the inability of the immune system to mount an effective immune response against head and neck squamous cell carcinoma (HNSCC) could be a result of the immunosuppression mediated through soluble factors that are secreted by tumour cells. Therefore, we investigated the effects of conditioned medium obtained from cultures of HNSCC cell lines (HNSCC-CM) on the function of dendritic cells (DC) and T cell immune response. In our study, we could not observe any inhibitory effect of HNSCC-CM on the maturation and the cytokine secretion pattern of DC. On the contrary, HNSCC-CM from two of three cell lines consistently decreased the quantity of IFN-gamma- and IL-4-secreting T cells upon restimulation in vitro. In conclusion, our data suggest that soluble factors secreted by HNSCC cells directly inhibit the function of effector T cells, rather than impeding the process of antigen presentation.

Keep on rolling: optimizing human islet transport conditions using a perfused rotary system.

The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process. ( 1) Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established.   Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused "rotary transport system for islets" termed "ROTi." Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating "standard" shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated "standard" shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.

EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis.

AIMS: Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer. MATERIAL AND METHODS: EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term 'EpCAM overexpression' was reserved for tissues showing a total immunostaining score >4. RESULTS: EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour. CONCLUSION: EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.

Challenging small human hepatocytes with opiates: further characterization of a novel prototype bioartificial liver.

Bioartificial liver (BAL) systems can take over liver functions in patients undergoing liver failure until transplantation. Recently, a novel prototype rotary BAL has been developed using small human hepatocytes (SH). This study investigated the metabolism of opiates morphine and methadone in the BAL and their influence on the basic cell culture parameters, viability, and growth of SH. Opiates may be present in patients due to pain therapy, anticancer treatment, or drug abuse. Cells were cultivated in the BAL for a total of 12 days and exposed twice to 100 microg/L of morphine or methadone. Morphine and methadone concentrations were analyzed using gas chromatography with a mass spectrometry detector. Further, the production of albumin, lactate dehydrogenase release, lactate release, urea production, and glucose consumption were measured. Cell viability and growth were determined by confocal microscopy. Cytochrome P 3A4 and uridindiphosphat (UDP) glucuronosyl transferase 2B7 in SH were analyzed by western blot. The mean cell density during treatment was 5.5 +/- 0.7 x 10(6) cells/mL (n = 6) and was not altered significantly by the opiates. Cell viability stayed above 90%. Morphine was not reduced by SH and was a stress factor as determined by decreased metabolic activity. On the other hand, SH metabolized methadone showing first-order kinetics: the first-order rate constant k = 0,019, half-life t(1/2) = 36 h. Methadone metabolism led to decreased urea and albumin production. The expression of cytochrome P 3A4, mainly responsible for methadone metabolism, was proved in SH. The prototype BAL is basically suited to support liver functions, provided patients receive therapy with methadone.

Introduction of a novel prototype bioartificial liver support system utilizing small human hepatocytes in rotary culture.

The aim of this study was to establish a stand-alone, perfused, rotary cell culture system using small human hepatocytes (SH) for bioartificial liver (BAL) support. SH were grown on cytodex 3 microcarriers (beads) to a maximum density of 1.2 +/- 0.3 x 10(7) cells per mL within 12 days. Size of aggregates formed by up to 15 beads was regulated by rotation speed. Cell function was proven by treatment with ammonia and galactose, and metabolism was analyzed. Treatment strategy was comprised of two phases, namely growth phase and treatment phase. Cells were grown for 6 days and subsequently incubated with ammonia or galactose for 2 days, followed by a 2-day regeneration period and another 2-day treatment phase. Consumption of glucose, release of lactate dehydrogenase, formation of lactate, and production of urea and albumin were determined regularly. Mean galactose consumption was 50 microg per 106 cells per hour, ammonia-induced urea formation was 3.6 microg per 106 cells per hour, and albumin production was 110 ng per 106 cells per hour. All metabolic parameters followed a logarithmic trend and were found to be very stable in the second half of the culture period when cells were treated with ammonia or galactose. Dissolved oxygen (%DO), pH, and temperature were monitored in-stream at intervals of 7 min, and the values were logged. Viability and morphology of cells were monitored via confocal microscopy. Viability was around 95% in controls and 90% during treatment. Promising results were obtained in support of our ongoing efforts to establish a fully autonomous BAL support device utilizing SH as a bridge to transplantation.

Survival signaling by C-RAF: mitochondrial reactive oxygen species and Ca2+ are critical targets.

Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca(2+). Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca(2+) levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca(2+), which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca(2+) overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca(2+) homeostasis.

Small human hepatocytes in rotary culture for treatment of alcohol addicts? A pilot study.

BACKGROUND: Current approaches to support alcohol addict and/or benzodiazepine-treated patients with liver failure include culturing human cells to take over basic metabolic functions for a certain time. METHODS: Small human hepatocytes (SH) were grown in a rotary cell culture system, and their potential to metabolize alcohol and the benzodiazepines oxazepam and diazepam was evaluated. Control experiments were performed with SV40-immortalized HEP cells and cell respective drug-free media. RESULTS: Our results show that SH in rotary culture are able to metabolize ethanol in reasonable amounts compared with evaporation controls (p<0.01). Moreover, SH are also able to metabolize oxazepam and diazepam which proves their ability to perform conjugation and the presence of functional cytochrome P450 enzymes. Basic metabolic activities such as glucose consumption, albumin and urea production are not significantly influenced by the drugs used, which is a precondition for clinical use of these cells. Significantly increased lactate dehydrogenase release indicates enhanced cell death in cultures of SH incubated with either ethanol (p<0.05) or diazepam (p<0.005), but stable viability at or above 90% suggests that cell proliferation is able to keep up with drug-induced cell death. CONCLUSION: Our preliminary study provides evidence that SH are basically suited to support alcohol-abusing and/or benzodiazepine-treated patients undergoing liver failure.

Bilirubin inhibits tumor cell growth via activation of ERK.

Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo, tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.

Pharmacodiagnostic value of the HER family in head and neck squamous cell carcinoma.

Two protooncogene products, EGFR (Her-1, c-erbB-1) and HER2 (Her-2/neu, c-erbB-2), have been reported to be frequently overexpressed in head and neck squamous cell carcinoma (HNSCC). In order to identify patients who may benefit from targeted cancer treatment for these two molecules, we determined the expression status of EGFR and HER2 in 129 HNSCC tumor specimens. Two pharmacodiagnostic kits (EGFR pharmDx and HercepTest) were used to identify HNSCC tumors that overexpress EGFR or HER2. Overexpression of EGFR was detected in 42.6% of the tumor specimens, while HER2 was only rarely expressed (overexpression was observed in just 3.1% of all cases). Given the necessity of new therapeutic modalities for patients suffering from HNSCC, treatment EGFR signaling inhibitors appears to be warranted, whereas therapeutic intervention with HER2 inhibitors seems to be inappropriate in this tumor type.