Correlation from undiluted vitreous cytokines of untreated central retinal vein occlusion with spectral domain optical coherence tomography.

PURPOSE: To correlate inflammatory and proangiogenic key cytokines from undiluted vitreous of treatment-naïve central retinal vein occlusion (CRVO) patients with SD-OCT parameters. METHODS: Thirty-five patients (age 71.1 years, 24 phakic, 30 nonischemic) underwent intravitreal combination therapy, including a single-site 23-gauge core vitrectomy. Twenty-eight samples from patients with idiopathic, non-uveitis floaterectomy served as controls. Interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF-A) levels were correlated with the visual acuity (logMar), category of CRVO (ischemic or nonischemic) and morphologic parameters, such as central macular thickness-CMT, thickness of neurosensory retina-TNeuro, extent of serous retinal detachment-SRT and disintegrity of the IS/OS and others. RESULTS: The mean IL-6 was 64.7pg/ml (SD ± 115.8), MCP-1 1015.7 ( ± 970.1), and VEGF-A 278.4 ( ± 512.8), which was significantly higher than the control IL-6 6.2 ± 3.4pg/ml (P=0.06), MCP-1 253.2 ± 73.5 (P<0.0000001) and VEGF-A 7.0 ± 4.9 (P<0.0006). All cytokines correlated highly with one another (correlation coefficient r=0.82 for IL-6 and MCP-1; r=0.68 for Il-6 and VEGF-A; r=0.64 for MCP-1 and VEGF-A). IL-6 correlated significantly with CMT, TRT, SRT, dIS/OS, and dELM. MCP-1 correlated significantly with SRT, dIS/OS, and dELM. VEGF-A correlated not with changes in SD-OCT, while it had a trend to be higher in the ischemic versus the nonischemic CRVO group (P=0.09). CONCLUSIONS: The inflammatory cytokines were more often correlated with morphologic changes assessed by SD-OCT, whereas VEGF-A did not correlate with CRVO-associated changes in SD-OCT. VEGF inhibition alone may not be sufficient in decreasing the inflammatory response in CRVO therapy.

[Cytokine determination from vitreous samples in retinal vascular diseases]. / Zytokinbestimmung aus Glaskörperproben bei retinalen Gefäßerkrankungen.

PURPOSE: The aim of this study was to determine cytokine levels from vitreous samples of treatment-naive patients with diabetic retinopathy (DRP), retinal vein occlusion (RVO) and exudative age-related macular degeneration (ARMD). METHODS: In this study 187 patients (median age 67 years, 101 males) were treated with a combined drug therapy including a 23-gauge core vitrectomy. Interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and intravitreal vascular endothelial growth factor (VEGF-A) levels were determined a using cytometric bead assay (CBA) and compared to those of the control group. RESULTS: Compared to the control group all diseases had significantly elevated cytokine levels, except VEGF in ARMD. In DRP samples of patients with diffuse diabetic macula edema (DME) higher VEGF-A and MCP-1 levels were found than in patients with focal DME. Ischemic DRP had higher VEGF levels than non-ischemic DRP. All measured cytokines were significantly higher in central retinal vein occlusion (CRVO) than in branch retinal vein occlusion (BRVO). CONCLUSIONS: Differences in intravitreal cytokine levels in DRP, RVO and ARMD could be demonstrated. The knowledge of depicted specific characteristic dysregulation of cytokines could allow more targeted future therapies.

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents.

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 µM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Selection of a highly invasive neuroblastoma cell population through long-term human cytomegalovirus infection.

The human cytomegalovirus (HCMV) is suspected to increase tumour malignancy by infection of cancer and/or stroma cells (oncomodulation). So far, oncomodulatory mechanisms have been attributed to the presence of HCMV and direct action of its gene products on cancer cells. Here, we investigated whether the prolonged presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. The neuroblastoma cell line UKF-NB-4 was long-term (200 passages) infected with the HCMV strain Hi91 (UKF-NB-4(Hi)) before virus eradication using ganciclovir (UKF-NB-4(HiGCV)). Global gene expression profiling of UKF-NB-4, UKF-NB-4(Hi) and UKF-NB-4(HiGCV) cells and subsequent bioinformatic signal transduction pathway analysis revealed clear differences between UKF-NB-4 and UKF-NB-4(Hi), as well as between UKF-NB-4 and UKF-NB-4(HiGCV) cells, but only minor differences between UKF-NB-4(Hi) and UKF-NB-4(HiGCV) cells. Investigation of the expression of a subset of five genes in different chronically HCMV-infected cell lines before and after virus eradication suggested that long-term HCMV infection reproducibly causes specific changes. Array comparative genomic hybridisation showed virtually the same genomic differences for the comparisons UKF-NB-4(Hi)/UKF-NB-4 and UKF-NB-4(HiGCV)/UKF-NB-4. UKF-NB-4(Hi) cells are characterised by an increased invasive potential compared with UKF-NB-4 cells. This phenotype was completely retained in UKF-NB-4(HiGCV) cells. Moreover, there was a substantial overlap in the signal transduction pathways that differed significantly between UKF-NB-4(Hi)/UKF-NB-4(HiGCV) and UKF-NB-4 cells and those differentially regulated between tumour tissues from neuroblastoma patients with favourable or poor outcome. In conclusion, we present the first experimental evidence that long-term HCMV infection can result in the selection of tumour cell populations with enhanced malignancy.

Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells.

Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3(r)Nutlin(10 µM), harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3(r)Nutlin(10 µM) cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3(r)Nutlin(10 µM) cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3(r)Nutlin(10 µM) cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3(r)Nutlin(10 µM) and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.

Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles.

Human serum albumin (HSA) nanoparticles represent promising drug carrier systems. Binding of cytostatics to HSA nanoparticles may diminish their toxicity, optimise their body distribution and/or may overcome multidrug resistance. In the present study, doxorubicin-loaded HSA nanoparticle preparations were prepared. Doxorubicin was loaded to the HSA nanoparticles either by adsorption to the nanoparticles' surfaces or by incorporation into the particle matrix. Both loading strategies resulted in HSA nanoparticles of a size range between 150nm and 500nm with a loading efficiency of 70-95%. The influence on cell viability of the resulting nanoparticles was investigated in two different neuroblastoma cell lines. The anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution. Based on these result a standard protocol for the preparation of doxorubicin-loaded HSA nanoparticles for further antitumoural studies was established.

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells.

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.