Self-priming determines high type I IFN production by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are responsible for the robust and immediate production of type I IFNs during viral infection. pDCs employ TLR7 and TLR9 to detect RNA and CpG motifs present in microbial genomes. CpG-A was the first synthetic stimulus available that induced large amounts of IFN-α (type I IFN) in pDCs. CpG-B, however, only weakly activates pDCs to produce IFN-α. Here, we demonstrate that differences in the kinetics of TLR9 activation in human pDCs are essential for the understanding of the functional difference between CpG-A and CpG-B. While CpG-B quickly induces IFN-α production in pDCs, CpG-A stimulation results in delayed yet maximal IFN-α induction. Constitutive production of low levels of type I IFN in pDCs, acting in a paracrine and autocrine fashion, turned out to be the key mechanism responsible for this phenomenon. At high cell density, pDC-derived, constitutive type I IFN production primes pDCs for maximal TLR responsiveness. This accounts for the high activity of higher structured TLR agonists that trigger type I IFN production in a delayed fashion. Altogether, these data demonstrate that high type I IFN production by pDCs cannot be simply ascribed to cell-autonomous mechanisms, yet critically depends on the local immune context.

Mucin-1 and its relation to grade, stage and survival in ovarian carcinoma patients.

BACKGROUND: Mucin-1 is known to be over-expressed by various human carcinomas and is shed into the circulation where it can be detected in patient's serum by specific anti-Mucin-1 antibodies, such as the tumour marker assays CA 15-3 and CA 27.29. The prognostic value of Mucin-1 expression in ovarian carcinoma remains uncertain. One aim of this study was to compare the concentrations of Mucin-1 in a cohort of patients with either benign or malignant ovarian tumours detected by CA 15-3 and CA 27.29. Another aim of this study was to evaluate Mucin-1 expression by immunohistochemistry in a different cohort of ovarian carcinoma patients with respect to grade, stage and survival. METHODS: Patients diagnosed with and treated for ovarian tumours were included in the study. Patient characteristics, histology including histological subtype, tumour stage, grading and follow-up data were available from patient records. Serum Mucin-1 concentrations were measured with ELISA technology detecting CA 15-3 and CA 27.29, Mucin-1 tissue expression was determined by immunohistochemistry using the VU4H5 and VU3C6 anti-Mucin-1 antibodies. Statistical analysis was performed by using SPSS 18.0. RESULTS: Serum samples of 118 patients with ovarian tumours were obtained to determine levels of Mucin-1. Median CA 15-3 and CA 27.29 concentrations were significantly higher in patients with malignant disease (p< 0.001) than in patients with benign disease.Paraffin-embedded tissue of 154 patients with ovarian carcinoma was available to determine Mucin-1 expression. The majority of patients presented with advanced stage disease at primary diagnosis. Median follow-up time was 11.39 years. Immunohistochemistry results for VU4H5 showed significant differences with respect to tumour grade, FIGO stage and overall survival. Patients with negative expression had a mean overall survival of 9.33 years compared to 6.27 years for patients with positive Mucin-1 expression. CONCLUSIONS: This study found significantly elevated Mucin-1 serum concentrations in ovarian carcinoma patients as compared to those women suffering from benign ovarian diseases. However, it needs to be noted that Mucin-1 concentrations in carcinoma patients showed a rather high variability. Results from immunohistochemistry indicate that Mucin-1 has a prognostic relevance in ovarian carcinomas when evaluating the expression by VU4H5 antibody.

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).

Estrogen receptor promoter methylation predicts survival in low-grade ovarian carcinoma patients.

PURPOSE: Ovarian carcinoma is the third most common gynecological cancer and only short recurrence-free survival and overall survival times are achieved. The role of the estrogen receptor expression is well studied in breast cancer and breast cancer cell lines. Patients with positive estrogen receptor expression have a lower risk for recurrence and a better overall survival. Previous studies have shown that ESR1 methylation influences ovarian cancer development and might thus play a role regarding prognosis of ovarian carcinoma. METHODS: A total of 75 patients were identified that were treated for ovarian carcinoma by debulking surgery and adjuvant standard chemotherapy. Isolation and bisulfite treatment of genomic DNA from serial sections of surgically resected ovarian carcinoma tissue was performed using commercially available kits. For the detection of methylated ESR1 promoter sequences, real-time methylation-specific PCR was used. RESULTS: Promoter methylation did not show a correlation between clinical-pathological data for all patients. However, within the subgroup of low-grade ovarian carcinoma patients and patients with an ovarian tumor of low malignant potential methylation of the ESR1 promoter inversely correlated with survival (p = 0.031). CONCLUSIONS: Although small numbers of ovarian carcinoma patients were analyzed, methylation status might be useful as a prognostic marker within the subgroup of low-grade ovarian carcinoma patients. Further studies should investigate a larger cohort and also address the use of demethylation agents with respect to improve patient's prognosis in this subgroup of ovarian carcinoma patients.

Replication-dependent potent IFN-alpha induction in human plasmacytoid dendritic cells by a single-stranded RNA virus.

Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-alpha induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-alpha induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-alpha induction in PDC. HRSV-induced IFN-alpha production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-alpha by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-alpha induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.