The use of tumor antigen loaded dendritic cells (DC) is one of the most promising approaches to induce a tumor specific immune response in vivo. Several strategies have been designed to load DC with tumor antigens. In this study, we investigated the delivery of in vitro transcribed RNA and plasmid DNA into monocyte-derived, ie non-proliferating human DC, using several nonviral transfection methods including electroporation and lipofection. Green fluorescent protein (GFP) was used as a reporter gene and influenza matrix protein 1 (M1) as a model antigen for HLA class I restricted antigen presentation. Using electroporation in combination with DNA or with RNA, up to 11% of DC were GFP-positive. Using liposomes as a vehicle for DNA transport up to 10% of the DC were GFP-positive. A significant

Citation: Dieckow J, Brandt W, Hattermann K, et al. CXCR4 and CXCR7 mediate TFF3-induced cell migration independently from the ERK1/2 signaling pathway. Invest Ophthalmol Vis Sci. 2016;57:56-65. DOI:10.1167/ iovs.15-18129 PURPOSE. Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors. METHODS.We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3-CXCR4/CXCR7 interaction were accomplished by cell culture-based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade. RESULTS.We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture-based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity.CONCLUSIONS. Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface.

Objective. Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage.Methods. We used reverse transcriptasepolymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritisaffected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay.Results. Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor ␣-and interleukin-1␤-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilagedegrading MMPs and increased chondrocyte apoptosis.Conclusion. In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

Background:Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer.Methods:For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated.Results:The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment.Conclusion:Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.