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.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phosphoinositide-dependent membrane recruitment. An interacting protein was identi®ed, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These ®ndings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by speci®c protein±protein interactions.

Cervical cancer is the principal cause of death due to cancer in women. Five-year survival rate ranges from 15-80%, depending on the extent of the disease. New predictive markers for relapse may increase survival rates by improving treatment of patients at high risk for relapse. The gene products of CDH1 and CDH13, namely E-cadherin and Hcadherin, play a key role in cell-cell adhesion. Inactivation of the cadherin-mediated cell adhesion system, caused by aberrant methylation, is a common finding in human cancers. To test the hypothesis that CDH1/CDH13 methylation is a prognostic marker in cervical cancer we determined the methylation status of CDH1/CDH13 in serum samples from 93 cervical cancer patients. Methylation analysis was carried out using MethyLight. Aberrant methylation of the 5 -region of CDH1 or CDH13 was observed in 43% (40 of 93) of the patients. Cervical cancer patients with unmethylated CDH1/ CDH13 in serum samples showed significantly better diseasefree survival in univariate and multivariate analysis. Median disease-free survival for CDH1/CDH13 methylation negative and positive patients was 4.3 years and 1.2 years, respectively. Our results suggest that detection of aberrant methylation of CDH1/CDH13 may be of potential use as a marker for selecting cervical cancer patients at high risk for relapse who could benefit from additional systemic therapy.

Purpose: Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease.Experimental Design: The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique.Results: Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P ‫؍‬ 0.04) and overall survival (P ‫؍‬ 0.02) in comparison with patients with methylated MYOD1.Conclusions: To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

Purpose: Cancer of the uterine cervix is an important cause of death in women worldwide. Pap smears as a tool for screening decreased the incidence and mortality of cervical cancer dramatically. This proof of principle study aimed to develop a potential tool for cervical screening using a test that can be applied by patients without visiting a physician and to increase the coverage rate, especially of the high-risk population with low socioeconomic status.Experimental Design: Human papillomavirus (HPV) DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens of 13, 31, and 11 patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer, respectively, collected on a tampon, was performed using PCRbased methods to detect invasive cervical cancer and study whether these changes are already present in the precursor lesions.Results: High-risk HPV DNA was present in 68 and 82% of patients with high-grade SIL and invasive cervical cancer. DNA methylation of the 11 genes tested increased with severity of the cervical lesion. Unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes was able to predict the presence of invasive cervical cancers: one of the two clusters formed contained 9 of 11 invasive cervical cancers, as well as two high-grade SILs.Conclusions: HPV DNA and DNA methylation analyzed in cervicovaginal specimens are able to predict invasive cervical cancers. To detect all high-grade SILs when applying this test, genes that become methylated earlier throughout cervical carcinogenesis have to be defined.

We report the induction and reduction of adenosine receptor A2a and A3 mRNAs, respectively, during maturation of human monocyte-derived dendritic cells. Adenosine, an immunomodulatory molecule, is unstable in vitro; therefore we tested a stable agonist, 5'-(N-ethylcarboxamido)-adenosine, to explore the effect of adenosine receptor activation on dendritic cell function. We clearly show that adenosine receptor engagement affects the migratory activity of dendritic cells in three distinct settings. In human skin explant culture experiments the emigration of epidermal and dermal dendritic cells was diminished by the addition of 5'-(N-ethylcarboxamido)-adenosine. In a murine contact hypersensitivity assay 5'-(N-ethylcarboxamido)-adenosine caused a reduction in the numbers of epidermal and dermal dendritic cells arriving in the draining lymph node. In a chemotaxis assay of human dendritic cells in response to macrophage inflammatory protein 3beta (MIP-3beta)/CCL19, adenosine caused a delay in transmigration. Expression of a number of molecules involved in dendritic cell migration (CCR5, MIP-3beta/CCL19, and MDR-1) was reduced. Importantly, all other features of dendritic cells tested--phenotype, antigen uptake, cytokine production, T cell activation, and the T cell subset induction--remained unchanged. Dendritic cells carry antigens from the periphery to secondary lymphoid organs, where initiation of immune responses occurs. Increased adenosine release may modulate immune responses by delaying the encounter of antigen-loaded dendritic cells with T cells.

Tacrolimus treatment reduces the ability of DC to stimulate T cells and the impaired production of DC-derived IP-10 (CXCL10) and IL-12 might play a role in the immunosuppressive action of tacrolimus.

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