In Silico Approach for Immunohistochemical Evaluation of a Cytoplasmic Marker in Breast Cancer.

Breast cancer is the most frequently diagnosed cancer in women and the second most common cancer overall, with nearly 1.7 million new cases worldwide every year. Breast cancer patients need accurate tools for early diagnosis and to improve treatment. Biomarkers are increasingly used to describe and evaluate tumours for prognosis, to facilitate and predict response to therapy and to evaluate residual tumor, post-treatment. Here, we evaluate different methods to separate Diaminobenzidine (DAB) from Hematoxylin and Eosin (H&E) staining for Wnt-1, a potential cytoplasmic breast cancer biomarker. A method comprising clustering and Color deconvolution allowed us to recognize and quantify Wnt-1 levels accurately at pixel levels. Experimental validation was conducted using a set of 12,288 blocks of m × n pixels without overlap, extracted from a Tissue Microarray (TMA) composed of 192 tissue cores. Intraclass Correlations (ICC) among evaluators of the data of 0.634 , 0.791 , 0.551 and 0.63 for each Allred class and an average ICC of 0.752 among evaluators and automatic classification were obtained. Furthermore, this method received an average rating of 4.26 out of 5 in the Wnt-1 segmentation process from the evaluators.

P2X(7) receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis.

Oligodendrocyte death and demyelination are hallmarks of multiple sclerosis (MS). Here we show that ATP signaling can trigger oligodendrocyte excitotoxicity via activation of calcium-permeable P2X(7) purinergic receptors expressed by these cells. Sustained activation of P2X(7) receptors in vivo causes lesions that are reminiscent of the major features of MS plaques, i.e., demyelination, oligodendrocyte death, and axonal damage. In addition, treatment with P2X(7) antagonists of chronic experimental autoimmune encephalomyelitis (EAE), a model of MS, reduces demyelination and ameliorates the associated neurological symptoms. Together, these results indicate that ATP can kill oligodendrocytes via P2X(7) activation and that this cell death process contributes to EAE. Importantly, P2X(7) expression is elevated in normal-appearing axon tracts in MS patients, suggesting that signaling through this receptor in oligodendrocytes may be enhanced in this disease. Thus, P2X(7) receptor antagonists may be beneficial for the treatment of MS.

System xc- and glutamate transporter inhibition mediates microglial toxicity to oligodendrocytes.

Elevated levels of extracellular glutamate cause excitotoxic oligodendrocyte cell death and contribute to progressive oligodendrocyte loss and demyelination in white matter disorders such as multiple sclerosis and periventricular leukomalacia. However, the mechanism by which glutamate homeostasis is altered in such conditions remains elusive. We show here that microglial cells, in their activated state, compromise glutamate homeostasis in cultured oligodendrocytes. Both activated and resting microglial cells release glutamate by the cystine-glutamate antiporter system xc-. In addition, activated microglial cells act to block glutamate transporters in oligodendrocytes, leading to a net increase in extracellular glutamate and subsequent oligodendrocyte death. The blocking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or the system xc- antiporter prevented the oligodendrocyte injury produced by exposure to LPS-activated microglial cells in mixed glial cultures. In a whole-mount rat optic nerve, LPS exposure produced wide-spread oligodendrocyte injury that was prevented by AMPA/kainate receptor block and greatly reduced by a system xc- antiporter block. The cell death was typified by swelling and disruption of mitochondria, a feature that was not found in closely associated axonal mitochondria. Our results reveal a novel mechanism by which reactive microglia can contribute to altering glutamate homeostasis and to the pathogenesis of white matter disorders.

Excitotoxic oligodendrocyte death and axonal damage induced by glutamate transporter inhibition.

Glutamate uptake is crucial to terminate glutamate signaling and to prevent excitotoxicity. The present study describes the expression of functional glutamate transporters GLAST and GLT-1 in oligodendrocytes by means of electrophysiology, uptake assays, and immunocytochemistry. Inhibition of glutamate uptake, both in oligodendrocyte cultures and in isolated optic nerves, increases glutamate levels and causes oligodendrocyte excitotoxicity, which is prevented by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptor antagonists. Furthermore, glutamate transporter inhibitors or antisense oligonucleotides applied onto the optic nerve in vivo lead to oligodendroglial loss, massive demyelination, and severe axonal damage. Overall, these results demonstrate that the integrity of oligodendrocytes and white matter depends on proper glutamate transporter function. Deregulated transporter activity may contribute to acute and chronic white matter damage.