Intravitreal vascular endothelial growth factor.

PURPOSE: To evaluate whether a specific pre-analytical stabilization regimen is needed for naïve vitreous taps to detect true values of intrinsic VEGF levels. METHODS: Fourteen consecutive patients with different vitreomacular pathologies without blood-retina-barrier breakdown were scheduled for standard 23-gauge three-port pars plana vitrectomy, and naïve vitreous taps were sampled at the beginning of each procedure. The extracted vitreous specimen was split; one half was immediately stored in a -20 °C freezer (unstabilized samples) and the other half was instantly stabilized with albumin (2.5 % final conc.), followed by arginine stabilization (1.25 M final conc.) and consecutively stored in a -20 °C freezer (stabilized samples). RESULTS: Intravitreal VEGF was detected in all 14 analyzed samples (100 %). VEGF levels were shown to be 46.5 pg/ml ± 62.3 pg/ml (MV ± SD; range: 5.99-232.3 pg/ml) in unstabilized, and 120.4 pg/ml ± 94.4 pg/ml (range: 42.9 pg/ml-289.6 pg/ml) in stabilized vitreous samples. Intravitreal VEGF levels in stabilized vitreous samples were on average 2.6-fold, and thus significantly higher than in unstabilized taps of same eyes (p = 0.001, Wilcoxon test). VEGF levels in stabilized vitreous samples can be up to 8.5 times higher than in corresponding unstabilized vitreous taps of same eyes (bootstrap analysis). Intravitreal VEGF levels in unstabilized samples correlate with those in stabilized vitreous taps (r = 0.594; p = 0.025; Pearson). CONCLUSIONS: An adequate pre-analytic stabilization regimen is needed to evaluate the most accurate intravitreal VEGF levels. This in turn will result in a better understanding of the physiological as well as pathological role of VEGF within the eye. Furthermore, knowing the true value of intravitreal VEGF levels will help to calculate the dosage of intravitrealy applied anti-VEGF drugs.

Fibrinolysis in normal vitreous liquid.

Plasmin is the key enzyme of fibrinolysis. Plasmin-alpha2-antiplasmin (PAP) complexes are biomarkers of fibrinolysis activation. The purpose of our investigation was to evaluate the activity of fibrinolysis in normal human eyes, that is in eyes without blood-retina barrier breakdown (BRB), which has not been investigated so far. Twenty-two vitreal samples were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy for macular pucker removal, macular hole closure or vitreal floater removal from the central vitreous body. These samples were immediately stabilized with human albumin (2.5% final conc.) and arginine (1.25 mmol/l final conc.) and subsequently frozen. Plasminogen was functionally determined in an ultra-sensitive pNA reaction after activation with streptokinase (100% = functional plasminogen in pooled normal citrated plasma). PAP concentrations were measured by enzyme immune assay (EIA). Intravitreal functional plasminogen exhibited to be 1 ±â€Š0.65% (range: 0.2-2.49%). PAP concentrations ranged at levels of 14 ±â€Š9ng/ml (range: 2-33 ng/ml). Pearson's correlation quotient between functional plasminogen and PAP revealed to be r equal to -0.27 (P = 0.221). No adverse events or serious side effects occurred. Sampling vitreous fluid at the beginning of a standard 23-gauge three-port pars plana vitrectomy is a well tolerated procedure. A strict stabilization procedure for extracted vitreous specimen is necessary to obtain activities and concentrations that are close to the true intraocular value. There is a basal intraocular fibrinolysis, a possible target for intravitreal pharmacological therapy.

Neurotrophin receptor expression in human primary retinoblastomas and retinoblastoma cell lines.

BACKGROUND: Neurotrophin receptor signaling regulates proliferation, differentiation and death of neuronal cells. Expression of Trk receptors has been implicated in the pathogenesis and prognosis of embryonal tumors, including neuroblastoma, nephroblastoma, and medulloblastoma. PROCEDURE: We analyzed TrkA, TrkB, TrkC, and p75 expression using semi-quantitative RT-PCR in 23 retinoblastomas and 8 retinoblastoma cell lines. Comparison of mRNA expression with clinical variables as well as the proliferation (PI) and apoptotic index (AI) of the tumor, was performed by Pearson correlation analysis and two-sample t-test. RESULTS: Almost all tumor samples and cell lines demonstrated high expression of all Trk receptors. Expression of TrkB and its ligand, BDNF, was most pronounced, suggesting TrkB to be the major Trk receptor involved in retinoblastoma biology. In contrast, p75 expression was substantially reduced in a subset of tumors and cell lines, in particular compared to its expression in normal retina. Tumors with infiltrative growth demonstrated significantly lower relative levels of TrkC expression than localized tumors (P = 0.004). High expression of TrkA was associated with a higher AI (P = 0.04), and high expression of TrkC was associated with a younger age of the patients (P = 0.03). Inhibition of Trk signaling by K252a resulted in marked growth inhibition of retinoblastoma cells in vitro. CONCLUSIONS: Our findings suggest a role for neurotrophin signaling in the biology of retinoblastoma. General Trk inhibitors are effective in decreasing growth rates of retinoblastoma cells in vitro, and should be evaluated in in vivo studies.

Allelic loss in a minimal region on chromosome 16q24 is associated with vitreous seeding of retinoblastoma.

In addition to RB1 gene mutations, retinoblastomas frequently show gains of 1q and 6p and losses of 16q. To identify suppressor genes on 16q, we analyzed 22 short tandem repeat loci in 58 patients with known RB1 mutations. A subset of tumors was also investigated by conventional and matrix comparative genomic hybridization. In 40 of 58 (69%) tumors, we found no loss of heterozygosity (LOH) at any 16q marker. LOH was detected in 18 of 58 (31%) tumors, including five with allelic imbalance at some markers. In one tumor LOH was only observed at 16q24. As the parental origin of allele loss was unbiased, an imprinted locus is unlikely to be involved. Analysis of gene expression by microarray hybridization and quantitative RT real-time PCR did not identify a candidate suppressor in 16q24. Cadherin 13 (CDH13), CBFA2T3, and WFDC1, which are candidate suppressors in other tumor entities with 16q24 loss, did not show loss of expression. In addition, mutation and methylation analysis showed no somatic alteration of CDH13. Results in all tumors with chromosome 16 alterations define a single minimal deleted region of 5.7 Mb in the telomeric part of 16q24 with the centromeric boundary defined by retention of heterozygosity for a single nucleotide variant in exon 10 of CDH13 (Mb 82.7). Interestingly, clinical presentation of tumors with and without 16q alterations was distinct. Specifically, almost all retinoblastomas with 16q24 loss showed diffuse intraocular seeding. This suggests that genetic alterations in the minimal deleted region are associated with impaired cell-to-cell adhesion.