Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy

PURPOSE. Advanced glycation end products (AGES) form irreversible cross- links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS. By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGES were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS. There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS. This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.

Advanced breast cancer incidence following population-based mammographic screening

Background: Breast cancer mortality is declining in many Western countries. If mammography screening contributed to decreases in mortality, then decreases in advanced breast cancer incidence should also be noticeable.
Patients and methods: We assessed incidence trends of advanced breast cancer in areas where mammography screening is practiced for at least 7 years with 60% minimum participation and where population-based registration of advanced breast cancer existed. Through a systematic Medline search, we identified relevant published data for Australia, Italy, Norway, Switzerland, The Netherlands, UK and the USA. Data from cancer registries in Northern Ireland, Scotland, the USA (Surveillance, Epidemiology and End Results (SEER), and Connecticut), and Tasmania (Australia) were available for the study. Criterion for advanced cancer was the tumour size, and if not available, spread to regional/distant sites.
Results: Age-adjusted annual percent changes (APCs) were stable or increasing in ten areas (APCs of -0.5% to 1.7%). In four areas (Firenze, the Netherlands, SEER and Connecticut) there were transient downward trends followed by increases back to pre-screening rates.
Conclusions: In areas with widespread sustained mammographic screening, trends in advanced breast cancer incidence do not support a substantial role for screening in the decrease in mortality.

Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappa B pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.

In vitro testing of Advanced JAX (TM) Bone Void Filler System: species differences in the response of bone marrow stromal cells to beta tri-calcium phosphate and carboxymethylcellulose gel

The Advanced JAX (TM) Bone Void Filler System (AJBVFS) is a novel bone graft material manufactured by Smith and Nephew Orthopaedics Ltd. and comprises beta tri-calcium phosphate granules with carboxymethylcellulose (CMC) gel as a handling agent. This study investigated the potential, in vitro, of the AJBVFS to function as a delivery system for cell therapy to enhance healing of bone defects. The attachment of rabbit bone marrow stromal cells (rbBMSCs), human BMSCs (hBMSCs) and human bone-derived cells (hBDCs) to JAX (TM) granules and the effect of CMC gel on cell proliferation and differentiation were investigated. There were slight species differences in the number and morphology of cells attached on the JAX (TM) granules with less rbBMSC attachment than human. All cells tolerated the presence of CMC gel and a reduction in cell number was only seen after longer exposure to higher gel concentrations. Low concentrations of CMC gel enhanced proliferation, alkaline phosphatase (ALP) expression and ALP activity in human cells but had no effect on rbBMSC. This study suggests that AJBVFS is an appropriate scaffold for the delivery of osteogenic cells and the addition of CMC gel as a handling agent promotes osteogenic proliferation and differentiation and is therefore likely to encourage bone healing.

A phase I study of the Heat Shock Protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced, solid tumours.

PURPOSE: A phase I study to define toxicity and recommend a phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response, and definition of a biologically effective dose (BED). PATIENTS AND METHODS: Patients with advanced solid cancers were treated with weekly, intravenous (i.v.) 17-DMAG. An accelerated titration dose escalation design was used. The maximum tolerated dose (MTD) was the highest dose at which = 1/6 patients experienced dose limiting toxicity (DLT). Dose de-escalation from the MTD was planned with mandatory, sequential tumor biopsies to determine a BED. Pharmacokinetic and pharmacodynamic assays were validated prior to patient accrual. RESULTS: Twenty-five patients received 17-DMAG (range 2.5-106 mg/m(2)). At 106 mg/m(2) of 17-DMAG 2/4 patients experienced DLT, including one treatment-related death. No DLT occurred at 80 mg/m(2). Common adverse events were gastrointestinal, liver function changes, and ocular. Area under the curve and mean peak concentration increased proportionally with 17-DMAG doses 80 mg/m(2) or less. In peripheral blood mononuclear cells significant (P