Time- and temperature-dependent stability of growth factor peptides in human autologous serum eye drops.

PURPOSES: To develop a step-by-step production method for human autologous serum (AS) eye drops that was broadly compliant with US Food and Drug Administration requirements for reinjection of processed biological substances. To determine optimum storage conditions for AS eye drops by measuring the concentration of growth factor peptides (GFP) as a function of storage temperature and storage duration. METHODS: AS derived from the blood of 3 healthy male volunteers was produced using a closed, vacuum-driven, cascade-filtration system under sterile, low-pyrogen conditions. In-process controls included methods for monitoring protein electrophoretic mobility and degradation rate and the content of free hemoglobin and endotoxin. Stability of transforming growth factor beta1, substance P, nerve growth factor, calcitonin gene-related peptide, insulin-like growth factor 1, and epidermal growth factor was evaluated at -15 degrees C, +4 degrees C, +25 degrees C, +37 degrees C, and +42 degrees C at different time intervals (hours to weeks). The main outcome measures were the concentrations of GFP, endotoxin, and lipid peroxidation by-products (a proxy measure for protein degradation) in dilute AS. RESULTS: The stability of GFP varies: transforming growth factor beta1, nerve growth factor, epidermal growth factor, and insulin-like growth factor 1 were more temperature and time resistant, but substance P and calcitonin gene-related peptide significantly degraded at +4 degrees C in 24 hours. Endotoxin and lipid peroxidation by-products were not significantly increased by processing. CONCLUSIONS: This pilot study developed a closed, cascade-filtration system that was an effective method for the production of high-quality, low-pyrogen AS. The processing method broadly complied with Food and Drug Administration requirements for reinjection of biological substances. Variable GFP stability was observed at +4 degrees C and above. For clinical use, AS should be packaged in daily-use containers, which should be stored frozen; the container in active use should be refrigerated between doses.

Serum growth factor analysis in dry eye syndrome.

BACKGROUND: To perform a comprehensive serum growth factor analysis in dry eye syndrome patients and to compare this with matched controls. METHODS: Six female dry eye syndrome patients and six age- and gender-matched controls were recruited. Whole blood was collected, allowed to clot and then centrifuged. Serum was extracted by using sterile technique. Enzyme-linked immunosorbent assays were performed to quantify serum growth factor levels. RESULTS: Levels of transforming growth factor-beta 1 and 2 (TGF-beta1 and beta2), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), acidic and basic fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA, AB and BB (PDGF-AA, AB and BB), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) were quantified, and statistical analysis was performed by using the Mann-Whitney U-test with the Bonferroni correction. CONCLUSIONS: No significant difference was found between serum growth factor levels in dry eye syndrome patients versus controls. Our study provides comprehensive analysis of serum growth factor levels in autologous serum eye drops produced from ocular surface disease patients. A knowledge of growth factor levels in serum may be important because of the increasing use of autologous serum eye drops in refractory ocular surface diseases and for an understanding of how topical serum may provide benefit.

Xenopus CDC7/DRF1 complex is required for the initiation of DNA replication.

The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.