Objective: The main objective of the study is to evaluate the unique signature of exosome-derived proteins as biomarkers for early detection of high-grade serous ovarian cancer (HGSOC).

Objective: TMEM205 is a novel transmembrane protein that may be associated with platinum resistance (PR) in high-grade serous ovarian cancer (HGSOC), but the specific mechanism associated with this resistance is still unknown. The goals of this study are to show that TMEM205 expression is linked with platinum-resistant HGSOC compared to platinum-sensitive and benign disease and to investigate the role of the TMEM205-CD1B (an exosome-related protein) axis in mediating this PR.

Objectives: To investigate how hypoxia facilitates the secretion of exosomes in the tumor microenvironment and how these exosomes contribute to tumor metastasis and drug resistance in ovarian cancer. Methods: Exosomes were obtained through ovarian cancer cell lines and human specimens. The cell lines cultures were performed in normoxic and hypoxic (1%) conditions. After institutional review board approval, ascites samples were obtained from patients undergoing laparotomy or paracentesis for ovarian cancer. Released exosomes were then isolated using Exo-quick. A nanoparticle tracking analyzer was used to quantify their size and concentration. Size was confirmed with transmission electron microscopy. Western-blotting was done to identify the exosomes with specific markers CD9, CD63, and EpCam and to confirm the presence of oncoproteins. We further analyzed the migratory potential of these exosomes in normal and cancer cell lines as well as with an in vivo orthotopic ovarian tumor model. Transfection was done to knock down STAT3 in ovarian cancer cell lines to elucidate its effect on exosome release. Results: Exosome concentration was increased 2- to 4-fold in hypoxic (vs normoxic) conditions. The knockdown of STAT3 in ovarian cancer cell lines reduced the release of exosomes in the setting of hypoxia. The hypoxic exosomes increased the migration potential of normal and cancer cell lines. In addition, these exosomes were found to carry the activated form of oncoproteins such as pSTAT3 and FAS. Ovarian cancer cell cultures performed in the presence of hypoxic exosomes and injected into mice showed more aggressive tumor growth and metastatic spread including prominent mesenteric nodules. In comparison, mice injected with ovarian cancer cells that underwent cultures with normoxic exosomes experienced tumor development at the primary site (ovary) of tumor injection, but had no evidence of metastasis. Conclusions: Hypoxia-mediated release of exosomes from ovarian cancer is influenced by STAT3. Hypoxic exosomes carry oncogenes and are associated with increased tumor burden and metastatic spread. These findings suggest that hypoxia-mediated exosome release facilitated by STAT3 may play an important role in the development of advanced ovarian cancer and serve as a target for therapeutic intervention.

Objectives: We tested a novel anticancer compound, HO-3867, on ovarian cancer in a clinically relevant, preclinical orthotopic mouse model by injecting tumor cells directly into the intrabursal space and allowing the tumor to grow in its native microenvironment. This model allows for the evaluation of efficacy in treating the primary tumor mass and permits investigation into the potential for prevention and/or treatment of metastatic tumor growth.

We have developed a coculture system which in parallel indicates the sensitizing and irritative potential of xenobiotics. The assay is named loose-fit coculture-based sensitization assay (LCSA) and may be performed within 5days. The system is composed of human monocytes that differentiate to a kind of dendritic cells by 2-day culturing in the presence of allogenic keratinocytes. The culture medium is enriched by a cocktail of recombinant cytokines. On day 3, concentration series of probes are added. On day 5, cells are harvested and analyzed for expression range of CD86 as a marker of sensitizing potential and for uptake of the viability stain 7-AAD as a marker of irritative potential. Estimation of the concentration required to cause a half-maximal increase in CD86 expression allowed quantification of sensitizing potential, and estimation of the concentration required to reduce viability to 50% allowed quantification of irritative potential. Examination of substances with known potential resulted in categorization of test scores. To evaluate our data, we have compared results with those of the validated animal-based sensitization test, the murine local lymph node assay (LLNA, OECD TG 429). To a large extent, results from LCSA and from LLNA achieved analogous grouping of allergens into categories like weak–moderate–strong. However, the new assay showed an improved capacity to distinguish sensitizers from non-sensitizers and irritants. In conclusion, the LCSA contains potential to fulfil the requirements of the EU's programme for the safety of chemicals “Registration, Evaluation, Authorisation and Restriction of chemical substances” (REACH, 2006) to replace animal models.

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

The cytokines IL-6, IL-1beta, TGF-beta, and IL-23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ-resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC-like cells TLR2-dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor gammabeta was detected. To clarify if IL-17(+)cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c(+)/langerin(+) monocyte-derived LC-like cells (MoLC), macrophage-activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL-6, IL-1beta, and IL-23. TGF-beta, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti-TLR2 inhibited secretion of IL-6, IL-1beta, and IL-23 by MoLC, while TGF-beta was unaffected. The amount of IL-17 and the ratio of IL-17 to IFN-gamma expression was higher in MoLC- than in monocyte-derived DC-cocultured Th cells. Anti-IL-1beta, -TGF-beta and -IL-23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN-stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

The cytokines IL-6, IL-1b, TGF-b, and IL-23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ-resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC-like cells TLR2-dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor cb was detected. To clarify if IL-17cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c/ langerin monocyte-derived LC-like cells (MoLC), macrophage-activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL-6, IL-1b, and IL-23. TGF-b, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti-TLR2 inhibited secretion of IL-6, IL-1b, and IL-23 by MoLC, while TGF-b was unaffected. The amount of IL-17 and the ratio of IL-17 to IFN-c expression was higher in MoLCthan in monocyte-derived DC-cocultured Th cells. Anti-IL-1b, -TGF-b and -IL-23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN-stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

Mercaptobenzothiazole (MBT) is used while manufacturing natural rubber products. Our study deals with assessing its allergenic potential following dermal and oral routes of exposure, using a biphasic local lymph node assay (LLNA). Female Balb/c mice were treated with MBT (dermally 3, 10, 30% concentrations in DMSO; orally 1, 10, 100 mg/kg doses in corn oil) on the back (dermal study) or through oral administration (oral study) on days 1-3 followed by auricular application of 3, 10 and 30% concentrations, respectively, on days 15-17. End points determined on day 19 included ear thickness, ear punch weight, lymph node weight, lymph node cell count, and lymphocyte subpopulations (CD4+, CD8+, CD45+). After dermal application of 3% or 10% solution, a significant increase in cell count and lymph node weight along with significant decrease in CD8+ cells was observed. After initial oral administration of 1 mg/kg, we noticed a significant amplification in cell count. Following oral administration of 10 mg/kg, we observed a similar increase in cell count and lymph node weight. The results of our study show that the modified biphasic LLNA protocol can be used to study the sensitising potential of a compound also following the oral route of exposure.

Establishing of alternatives to animal tests is ethically desirable and gains in importance in context of new European Union regulations such as REACH. We have refined our new in vitro assay for prediction of the sensitizing potency of xenobiotics. Monocytes cocultured with primary human keratinocytes develop to a novel class of in vitro generated dendritic cells after treatment with transforming growth factor beta and Interleukin-4 in serum-free medium. These dendritic cell-related cells (DCrc) are the key players in the loose-fit coculture-based sensitization assay (LCSA). Assay duration and cytokine consumption could be cut down without impairing the assay's functionality. DCrc showed a dose-dependent upregulation of CD86 after treatment with the contact allergens 2,4,6-trinitrobenzenesulfonic acid, the prohapten isoeugenol, and alpha-hexyl cinnamic aldehyde. The metal allergens nickel and cobalt could be detected by measuring Interleukin-6 and macrophage inflammatory protein 1-beta (MIP-1beta, CCL-4) in coculture supernatants. The irritant zinc elicited no reaction. Lipopolysaccharide produced upregulation of CD86, IL-6 and MIP-1beta. Determination of tolerable concentrations of an allergen in consumer products requires a widely accepted sharp quantitative assay. Animal-based assays do not meet this requirement. The LCSA provides dose-response information, thereby allowing prediction of the relative ability of a substance to induce sensitization.

We are developing a new, animal-free assay for determination of the sensitizing potential of a substance. The design of this assay is based on current immunological knowledge of the pathogenesis of allergic contact dermatitis. It integrates human dendritic cells and keratinocytes, which are both known to be critically involved in vivo. The read-out system uses molecular responses typically occurring after an encounter of the skin with contact allergens. The assay provides concentration-response information, by which the relative ability of a chemical to induce sensitization can be predicted. Additionally, the assay defines the border-concentration of general toxicity of a substance. Weak allergens and even prohaptens are detectable. We have called the assay LCSA, loose-fit coculture-based sensitization assay.

In epidermal Langerhans cells (LCs), the expression pattern and the functions of TLRs have been poorly characterized. By using mAb, we show that LCs from human skin express TLR1, -2, -5, -6, and -9, the cognate receptors for detection of specific bacteria-derived molecules. As compared with other TLR agonists, LCs acquired a more matured phenotype when activated by specific bacterial or synthetic TLR2 agonists. In addition, monocyte-derived Langerin(+)/CD1c(+)LCs (CD1c(+)MoLCs) secreted higher amounts of IL-6 and TNF-beta by stimulation via TLR2 than by stimulation via TLR3, -4, -5, -8, and -9. In contrast to MoLCs, dendritic cells, generated from the same donor monocytes, were activated by agonists of TLRs other than TLR2 as well. Lipopeptides triggering TLR2 induced IL-1R-associated kinase-1 phosphorylation and migration toward the chemokines CCL19 and CCL21 in epidermal LCs and CD1c(+)MoLCs. Up-regulation of CD86, CD83, and CCR7, TNF-alpha and IL-6, and NF-kappa B activation and proliferation of CD4(+)T cells could be inhibited TLR2-specific blockage using antibodies prior to TLR2 activation. Application of anti-TLR1, anti-TLR6, and anti-TLR2 indicated an exclusive role of TLR2 in IL-6 induction in human LCs. Collectively, our results show that TLR2 expressed by LCs mediates inflammatory responses to lipopeptides, which implicates a central role in sensing pathogens in human skin.

Background Neurotrophins are involved in inflammatory reactions influencing several cells in health and disease including allergy and asthma. Dendritic cells (DCs) play a major role in the induction of inflammatory processes with an increasing role in allergic diseases as well.Objective The aim of this study was to investigate the influence of neurotrophins on DC function.Methods Monocyte-derived dendritic cells were generated from allergic and non-allergic donors. Neurotrophin receptors were demonstrated by western blotting, flow cytometry and fluorescence microscopy. Activation of small GTPases was evaluated by pull-down assays. DCs were incubated with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and supernatants were collected for measurement of IL-4, IL-6, IL-10, IL-12p70, TNF-alpha and TGF-beta.Results Receptor proteins were detectable by western blot, fluorescence activated cell sorting analysis and fluorescence microscopy. Signalling after neurotrophin stimulation occurred in a ligand-specific pattern. NGF led to decreased RhoA and increased Rac activation, while BDNF affected RhoA and Rac activity in a reciprocal fashion. Cells of allergics released a significantly increased amount of IL-6, while for healthy subjects a significantly higher amount of IL-10 was found.Conclusion These data indicate that DCs are activated by the neurotrophins NGF and BDNF by different pathways in a receptor-dependant manner. These cells then may initiate inflammatory responses based on allergic sensitization releasing preferred cytokines inducing tolerance or a T-helper type 2 response.

Protection against contact allergy begins with the collection of reliable data about the sensitizing potential of chemicals. Today, the local lymph node assay (LLNA) in mice is widely used to identify sensitizing substances. For several reasons, an in vitro assay could be preferable to animal experiments. We propose an in vitro test for the detection of a sensitizing potential of a chemical composed of a single layer of human nondifferentiating keratinocytes and of allogenic floating monocytes which are cocultured in serum‐free medium in the presence of a cytokine cocktail. Within days, the coculture develops to an allergen‐ sensitive system consisting of activated keratinocytes and of mobile dendritic cell‐related cells (DC‐related cell). The sensitizing potential can be determined by analyzing the expression of the dendritic cell maturation marker CD86. For the model contact allergens tested so far [trinitrobenzenesulfonic acid (TNBS), phenylendiamine, and 4‐aminoacetanilide], the strength of the reaction was in concordance with results from the LLNA. Sensitivity of the assay allowed testing at concentrations without general cytotoxicity. Thus, a differentiation between allergens and irritants was possible. Regarding cytokine secretion, the assay distinguished between the allergen TNBS and the Toll‐like receptor ligand lipopolysaccharide. The coculture can be set up from cryopreserved cells. The assay is easy to perform and reproducible. Donor‐variance is negligible. This in vitro assay based on a loose‐fit coculture is a reasonable approach to screen for the sensitizing potential of xenobiotics and might partially replace the LLNA and other animal tests.

BACKGROUND:Neurotrophins are produced by various cells upon different stimuli and participate in the initiation and regulation of inflammation in various diseases including allergy and asthma, but little is known about the production and control of neurotrophins by dendritic cells (DCs). The aim of this study was to assess whether DCs produce the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and whether inflammatory stimuli or allergens are able to induce the production of neurotrophic factors.METHODS:Monocyte-derived dendritic cells (MoDCs) were generated from different donors. The neurotrophins NGF and BDNF were demonstrated by RT-PCR, Western blotting, flow cytometry analysis and fluorescence microscopy. MoDCs were cultured and stimulated with lipopolysaccharide (LPS) or allergen for 24 h. The supernatants and cells were collected. Measurement for NGF and BDNF was performed by ELISA.RESULTS:DCs express mRNA for the neurotrophins NGF and BDNF. Proteins were detectable by Western blot, FACS analysis and fluorescence microscopy. LPS led to an up-regulation of BDNF, while NGF was unaffected. Cell lysates demonstrated an increased amount of BDNF after stimulation with LPS or allergen, while NGF was not affected significantly.CONCLUSIONS:DCs are a source of neurotrophins. LPS selectively regulates the production of BDNF. Allergen stimulation leads to an LPS-independent regulation. This contributes to a complex involvement of neurotrophins in allergic diseases.

Natural regulatory CD4(+)CD25(+)Foxp3(+) T cells control peripheral immune responses. Freshly isolated regulatory T-cell populations are regarded as being unable to suppress the proliferation of strongly stimulated effector T cells. We now provide evidence that it is not the strength of the proliferative signal to effector T cells but activation and accessibility of regulatory T cells that determine whether suppression may occur. Human regulatory T cells were initially cocultured with allogeneic monocyte-derived dendritic cells for a short time and were then rendered accessible for effector T cells by removal of the dendritic cells. That way activated regulatory T cells effectively suppressed the proliferation of autologous effector T cells which was strongly driven by cell-sized Dynabeads coated with CD3/CD28 antibodies. Although regulatory T cells are known to display MHC II molecules and to upregulate their expression along with activation, a role of MHC II molecules in forming the contact to effector T cells was not yet envisaged. However, blocking of MHC II on activated regulatory T cells abrogated their suppressive potential. It should not be excluded that self-MHC molecules on physically accessible activated regulatory T cells arrange the contact to effector T cells.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG exhibit their immunostimulatory activities by binding to TLR. Here, we show that human monocyte-derived dendritic cells (moDC) contain TLR9 protein, surprisingly, in amounts comparable with plasmacytoid DC (pDC). Immature moDC but not mature moDC nor monocytes captured CpG-ODNs. moDC stimulation with the CpG-A ODN D19 up-regulated CD83, CD86, and HLA-DR. Without CD40 ligand costimulation, full maturation was not achieved. D19-stimulated moDC primed allogeneic CD4(+)-T cells for proliferation and differentiation into IFN-gamma-secreting Th1 cells. Neither IL-12 nor IL-6 or TNF-alpha was involved. Microarray analysis pointed to a participation of Type I IFNs. In fact, D19-stimulated moDC secreted considerable amounts of IFN-alpha. This indicates that moDC themselves sense viral and bacterial DNA and do not need help from pDC.