CXCL16 and oxLDL are induced in the onset of diabetic nephropathy

Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated α3-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored α3 integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN

L1-CAM expression in ccRCC correlates with shorter patients survival times and confers chemoresistance in renal cell carcinoma cells

Conflicting data exist about the expression of L1 cell adhesion molecule (L1-CAM) in clear cell renal cell carcinoma (ccRCC). To determine the clinical usefulness of L1-CAM as a therapeutic or prognostic marker molecule in renal cancer patients, we analyzed its expression on a cohort of 282 renal cell carcinoma (RCC) patients. L1-CAM expression was found in 49.5% of 282 renal cancer tissues. Importantly, L1-CAM expression in patients with ccRCC was associated with significantly shorter patient survival time. We further present evidence that L1-CAM was involved in the resistance against therapeutic reagents like rapamycin, sunitinib and cisplatin. The downregulation of L1-CAM expression decreased renal cancer cell proliferation and reduced the expression of cyclin D1. In addition, we found out that Von Hippel-Lindau (VHL) deficiency was accompanied by a downregulation of the transcription factor PAX8 and L1-CAM. In normal renal tissue, PAX8 and L1-CAM were co-expressed in collecting duct cells. Importantly, the downregulation of PAX8 by small interfering RNA increased the expression of L1-CAM and concomitantly induced the migration of renal cancer cells. Furthermore, we observed in 65.3% of 282 RCC patients a downregulation of PAX8 expression. With chromatin immunoprecipitation analysis, we additionally demonstrate that PAX8 can bind to the promoter of L1-CAM and we further observed that the downregulation of PAX8 was accompanied by increased L1-CAM expression in a high fraction of ccRCC patients. In summary, we show that VHL and PAX8 are involved in the regulation of L1-CAM in renal cancer and L1-CAM represents an important therapeutic and prognostic marker protein for the treatment of ccRC

ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

<p>Abstract</p> <p>Background</p> <p>The importance of the Notch signaling in the development of glomerular diseases has been recently described. Therefore we analyzed in podocytes the expression and activity of ADAM10, one important component of the Notch signaling complex.</p> <p>Methods</p> <p>By Western blot, immunofluorescence and immunohistochemistry analysis we characterized the expression of ADAM10 in human podocytes, human urine and human renal tissue.</p> <p>Results</p> <p>We present evidence, that differentiated human podocytes possessed increased amounts of mature ADAM10 and released elevated levels of L1 adhesion molecule, one well known substrate of ADAM10. By using specific siRNA and metalloproteinase inhibitors we demonstrate that ADAM10 is involved in the cleavage of L1 in human podocytes. Injury of podocytes enhanced the ADAM10 mediated cleavage of L1. In addition, we detected ADAM10 in urinary podocytes from patients with kidney diseases and in tissue sections of normal human kidney. Finally, we found elevated levels of ADAM10 in urinary vesicles of patients with glomerular kidney diseases.</p> <p>Conclusions</p> <p>The activity of ADAM10 in human podocytes may play an important role in the development of glomerular kidney diseases.</p

PAX2 Regulates ADAM10 Expression and Mediates Anchorage-Independent Cell Growth of Melanoma Cells

PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma

Junctional adhesion molecule-A (JAM-A) is one component of tight junctions which are involved in important processes like paracellular permeability, cell polarity, adhesion, migration, and angiogenesis. Here we describe JAM-A expression in distal convoluted tubule, connecting tubule, and in cells of the collecting duct of the healthy human kidney. In addition, JAM-A was weakly expressed in cells of the proximal tubule. Using immunofluorescence, FACS and Western blot analysis we investigated JAM-A expression in tubular cells in vitro. Interestingly, treatment of HK-2 cells with IFN-gamma and TNF-alpha resulted in a metalloproteinase mediated downregulation of JAM-A. Importantly, in a tissue micro-array JAM-A protein expression was significantly downregulated in patients with clear cell renal cell carcinoma. Furthermore, knockdown of JAM-A with JAM-A specific siRNA induced the migration of RCC4 cells. In summary, downregulation of JAM-A is an early event in the development of renal cancer and increases the migration of renal cancer cells

Downregulation of PAX2 decreases the proliferation, migration and invasion of melanoma cells.

<p>Anchorage-dependent (<b>A</b>) and anchorage-independent (<b>B</b>) cell growth was investigated by using a MTT proliferation assay. Twenty-four hours after siRNA transfection, SkMel5 cells treated with transfection reagents alone (mock) or transfected with scrambled siRNA (sc-siRNA) or PAX2-specific siRNAs were seeded into uncoated anchorage dependent cell growth) or polyHEME coated (anchorage independent cell growth) 96 well plates and cell growth was measured 24, 48 and 72 hours later using a MTT-assay. 3 independent experiments have been performed and statistical analysis has been performed using Anova post-hoc analysis. ***P<0.001 considered statistically significant compared to control transfected cells (Mock). <b>###</b>P<0.001 considered statistically significant compared to scrambled-siRNA transfected cells, *P<0.01 considered statistically significant compared to scrambled-siRNA transfected cells. (<b>C</b>) Migration assay of SkMel5 cells was performed 48 h after the transfection with control siRNA (sc-siRNA) or PAX2 specific siRNA (PAX2-siRNA). ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA). (<b>D</b>) The invasive capacity of SkMel5 cells was analyzed 48 h after the transfection of contol (sc-siRNA) or PAX2 siRNA (PAX2-siRNA) in an invasion assay as described under material and methods ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA).</p

ADAM10 and PAX2 are co-expressed in melanocytes and melanoma cells in tissue sections of benign nevi and malignant melanoma.

<p>To determine if ADAM10 and PAX2 are co-expressed in melanocytes of benign nevi or in melanoma cells of patients with malignant melanoma, double immunofluorescence analysis on tissue sections has been performed. ADAM10 (<b>green</b>) and PAX2 (<b>red</b>) expression is detectable in melanocytes of benign nevi (<b>A</b> insets represent higher magnification of the single channels and the merged image of all 3 channels) and in melanoma cells of patients with malignant melanoma (<b>B</b> and <b>C</b> insets represent higher magnification of the single channels and the merged image of all 3 channels).</p

PAX2 and ADAM10 expression in melanocytes, keratinocytes and melanoma cells.

<p>(<b>A</b>) Western Blot analysis was performed to determine the PAX2 and ADAM10 expression in melanocytes (Mel43), keratinocytes and melanoma cells (A375, G361, IPC298, MeWo, NW1539 and SKMel5). Notably, 5 of 6 melanoma cell line show PAX2 and ADAM10 expression. β-Actin Western Blot analysis was performed to control equal protein loading. (<b>B</b>) Immunofluorescence staining of primary melanocytes Mel43 (left image) and the melanoma cell lines IPC298 (middle image) and G361 (right image) was performed to investigate the localisation of ADAM10 and PAX2. Cells were incubated with monoclonal ADAM10 and polyclonal PAX2 specific antibodies, followed by Alex488 coupled secondary antibodies (green) and Cy3 coupled secondary antibodies (red). The cells were stained with DAPI to visualize nuclei (blue). (<b>C</b>) The relative immunofluorescence intensity of ADAM10 and PAX2 expression in the melanocytes Mel43 and the melanoma celllines IPC298 and G631 were determined and depicted in a graph. ***P<0.001 PAX2 immunofluorescence intensity considered statistically significant compared to the PAX2 immunofluorescence intensity of melanocytes. <b>###</b>P<0.001 ADAM10 immunofluorescence intensity considered statistically significant compared to ADAM10 immunofluorescence intensity. (<b>D</b>) The specificity of ADAM10 and PAX2 immunofluorescence staining was controlled by using isotype specific control (control IgG) antibodies.</p

PAX2 regulates ADAM10 expression in melanoma cells.

<p>(<b>A</b>) Chromatin immunoprecipitation (ChIP) assay was performed with SKMel5 cells as described in material and methods. One representative experiment of three independently performed experiments is shown. (<b>B</b>) SKMel5 cells were transfected with pcDNA3.1 plasmid alone or with PAX2-pcDNA3.1 plasmid DNA. The expression of PAX2 and ADAM10 was determined by Western Blot analysis. β-actin was used to determine equal protein loading. (<b>C</b>) SkMel5 were transfected with 10 nM scrambled siRNA (sc-siRNA) or with 10 nM of two different PAX2-siRNAs (PAX2-siRNA1-2). 48 hours and 72 hours after the transfection, cells were lysed and the protein expression level of PAX2 and ADAM10 was investigated by Western Blot analysis. β-actin was used to determine equal protein loading. (<b>D</b>) SkMel5 were transfected with 10 nM scrambled siRNA (sc-siRNA) or with 10 nM of two different PAX2-siRNAs (PAX2-siRNA1-2). 48 hours and 72 hours after the transfection, cells were lysed and the protein expression level of PAX8 was investigated by Western Blot analysis. β-actin was used to determine equal protein loading. (<b>E</b>) Immunofluorescence analysis with ADAM10 and PAX2 specific antibodies were performed in sc-siRNA (left image) and PAX2- siRNA (right image) transfected SkMel-5 cells. ADAM10 expression was visualized by Cy3 coupled goat anti-mouse secondary antibodies, whereas PAX2 expression was detected with Alexa488 coupled goat anti-rabbit antibodies. (<b>F</b>) In the graphs the quantification of ADAM10 and PAX2 immunofluorescence intensity is shown. ***P<0.001 considered statistically significant compared to the sc-siRNA transfected SkMel5 cells.</p