Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH).

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. METHODS: The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark(R) XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatase (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. RESULTS: Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 - 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 - 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 - 1.0000). CONCLUSION: Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

APP and PS-1 mutations induce brain oxidative stress independent of dietary cholesterol: implications for Alzheimer's disease.

Epidemiological and biochemical studies strongly implicate a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). Mutation in the PS-1 and APP genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. The AD brain is under significant oxidative stress, including protein oxidation and lipid peroxidation. In the present study, protein oxidation and lipid peroxidation were compared in the brain homogenates from knock-in mice expressing mutant human PS-1 and APP in relation to the intake of dietary cholesterol. The APP and PS-1 mice displayed increased oxidative stress as measured by protein oxidation and lipid peroxidation, independent of dietary cholesterol. These results are discussed with reference to proposed therapeutic strategies of AD.

Chronic brain inflammation leads to a decline in hippocampal NMDA-R1 receptors.

BACKGROUND: Neuroinflammation plays a prominent role in the progression of Alzheimer's disease and may be responsible for degeneration in vulnerable regions such as the hippocampus. Neuroinflammation is associated with elevated levels of extracellular glutamate and potentially an enhanced stimulation of glutamate N-methyl-D-aspartate receptors. This suggests that neurons that express these glutamate receptors might be at increased risk of degeneration in the presence of chronic neuroinflammation. METHODS: We have characterized a novel model of chronic brain inflammation using a slow infusion of lipopolysaccharide into the 4th ventricle of rats. This model reproduces many of the behavioral, electrophysiological, neurochemical and neuropathological changes associated with Alzheimer's disease. RESULTS: The current study demonstrated that chronic neuroinflammation is associated with the loss of N-methyl-D-aspartate receptors, as determined both qualitatively by immunohistochemistry and quantitatively by in vitro binding studies using [3H]MK-801, within the hippocampus and entorhinal cortex. CONCLUSION: The gradual loss of function of this critical receptor within the temporal lobe region may contribute to some of the cognitive deficits observed in patients with Alzheimer's disease.

Attenuation of chronic neuroinflammation by a nitric oxide-releasing derivative of the antioxidant ferulic acid.

Chronic neuroinflammation and oxidative stress contribute to the neurodegeneration associated with Alzheimer's disease and represent targets for therapy. Ferulic acid is a natural compound that expresses antioxidant and anti-inflammatory activities. Nitric oxide is also a key modulator of inflammatory responses. Grafting a nitric oxide-releasing moiety onto anti-inflammatory drugs results in enhanced anti-inflammatory activity. We compared the effectiveness of ferulic acid with a novel nitric oxide-releasing derivative of ferulic acid in an animal model of chronic neuroinflammation that reproduces many interesting features of Alzheimer's disease. Lipopolysaccharide was infused into the 4th ventricle of young rats for 14 days. Various doses of ferulic acid or its nitric oxide-releasing derivative were administered daily. Both drugs produced a dose-dependent reduction in microglia activation within the temporal lobe. However, the nitric oxide-releasing ferulic acid derivative was significantly more potent. If we delayed the initiation of therapy for 14 days, we found no reduction in microglial activation. In addition, both drugs demonstrated antioxidant and hydroxyl radical scavenging abilities in in vitro studies. Overall, our results predict that a treatment using nitric oxide-releasing ferulic acid may attenuate the processes that drive the pathology associated with Alzheimer's disease if the treatment is initiated before the neuroinflammatory processes can develop.

The influence of brain inflammation upon neuronal adenosine A2B receptors.

Alzheimer's disease (AD) is associated with glial activation and increased levels of pro-inflammatory cytokines. Epidemiological results suggest that anti-inflammatory therapies can slow the onset of AD. Adenosine, acting at type-2 receptors, is an effective endogenous anti-inflammatory agent that can modulate inflammation both in the periphery and the brain. We investigated changes in the expression of adenosine type-2B (A2B) receptors and a related intracellular second messenger during chronic brain inflammation and following treatment with the non-steroidal anti-inflammatory drug flurbiprofen and its nitric oxide (NO)-donating derivative, HCT1026. Chronic infusion of lipopolysaccharide (LPS) into the 4th ventricle of young rats induced brain inflammation that was associated with microglial activation and reduced neuronal immunoreactivity for adenosine A2B receptors in the cortex. Daily administration of HCT1026, but not flurbiprofen, reduced microglial activation, prevented the down-regulation of A2B receptors and elevated tissue levels of cAMP. The results suggest that a therapy using an NO-releasing NSAID might significantly attenuate the processes that drive the pathology associated with AD and that this process may involve the activation of adenosine A2B receptors.

A nitric oxide-donating flurbiprofen derivative reduces neuroinflammation without interacting with galantamine in the rat.

Alzheimer's disease is associated with glial activation and increased levels of the cytokines as well as impaired forebrain cholinergic function. Current therapies focus on enhancing cholinergic function by administrating acetylcholinesterase inhibitors, such as galantamine. Epidemiological results also suggest that anti-inflammatory therapies might be effective in slowing the onset of the symptoms of Alzheimer's disease. The current study investigated the ability of a nitric oxide (NO)-donating derivative of the nonsteroidal anti-inflammatory drug (NSAID) flurbiprofen, HCT1026, to reduce brain inflammation in young rats. Inflammation was produced by chronic infusion of lipopolysaccharide (LPS) into the 4th ventricle. The release of NO from HCT1026 requires the action of esterase enzymes. The current study determined whether the effectiveness of HCT1026 was attenuated by simultaneous treatment with the acetylcholinesterase inhibitor galantamine. Daily administration of the HCT1026 significantly reduced microglial activation and these effects were not attenuated by galantamine therapy.

Beta-amyloid deposition in the brains of rats chronically infused with thiorphan or lipopolysaccharide: the role of ascorbic acid in the vehicle.

Beta-amyloid deposition and neuroinflammation are two important features of Alzheimer's disease (AD) that may influence its progression. Chronic infusion of lipopolysaccharide (LPS) into the rodent 4th ventricle reproduces many of the neurobiological changes seen in AD. Chronic infusion of ascorbic acid containing thiorphan, an inhibitor of the enzyme neprilysin that catabolizes beta-amyloid, into the hippocampus induces extracellular deposition of beta-amyloid fibrils. We investigated whether the combined presence of chronic neuroinflammation could exacerbate the deposition of beta-amyloid induced by thiorphan. The infusion of any solution containing ascorbic acid alone or with thiorphan or LPS increased the level of intraneuronal beta-amyloid immunoreactivity. Solutions that did not contain ascorbic acid were not associated with increased intraneuronal beta-amyloid immunoreactivity. The role of neprilysin in the deposition of beta-amyloid in AD brains remains undetermined.