We established microRNA profiles from active and inactive multiple sclerosis lesions. Using laser capture microdissection from multiple sclerosis lesions to pool single cells and in vitro cultures, we assigned differentially expressed microRNA to specific cell types. Astrocytes contained all 10 microRNA that were most strongly upregulated in active multiple sclerosis lesions, including microRNA-155, which is known to modulate immune responses in different ways but so far had not been assigned to central nervous system resident cells. MicroRNA-155 was expressed in human astrocytes in situ, and further induced with cytokines in human astrocytes in vitro. This was confirmed with astrocyte cultures from microRNA-155-|-lacZ mice. We matched microRNA upregulated in phagocytically active multiple sclerosis lesions with downregulated protein coding transcripts. This converged on CD47, which functions as a 'don't eat me' signal inhibiting macrophage activity. Three microRNA upregulated in active multiple sclerosis lesions (microRNA-34a, microRNA-155 and microRNA-326) targeted the 3'-untranslated region of CD47 in reporter assays, with microRNA-155 even at two distinct sites. Our findings suggest that microRNA dysregulated in multiple sclerosis lesions reduce CD47 in brain resident cells, releasing macrophages from inhibitory control, thereby promoting phagocytosis of myelin. This mechanism may have broad implications for microRNA-regulated macrophage activation in inflammatory diseases.
Extracellular matrix (ECM) proteins can modify immune reactions, e.g. by sequestering or displaying growth factors and by interacting with immune and glial cells. Here we quantified by quantitative polymerase chain reaction (qPCR) expression of 50 ECM components and 34 ECM degrading enzymes in multiple sclerosis (MS) active and inactive white matter lesions. COL1A1, COL3A1, COL5A1 and COL5A2 chains were induced strongly in active lesions and even more in inactive lesions. These chains interact to form collagen types I, III and V, which are fibrillar collagens. Biglycan and decorin, which can decorate fibrillar collagens, were also induced strongly. The fibrillar collagens, biglycan and decorin were largely found between the endothelium and astrocytic glia limitans in the perivascular space where they formed a meshwork which was closely associated with infiltrating immune cells. In active lesions collagen V was also seen in the heavily infiltrated parenchyma. Fibrillar collagens I and III inhibited in vitro human monocyte production of CCL2 (MCP-1), an inflammatory chemokine involved in recruitment of immune cells. Together, ECM changes in lesions with different activities were quantified and proteins forming a perivascular fibrosis were identified. Induced fibrillar collagens may contribute to limiting enlargement of MS lesions by inhibiting the production of CCL2 by monocytes.
Treatment options for most nervous system tumors remain limited and patients are often confronted with significant morbidity and reduced life expectancy. However, significant efforts are underway to find more effective therapies for patients with primary and secondary brain tumors. As more and more clinical trials for nervous system tumors are being conducted, it is increasingly important to optimize the conduct of clinical trials in neuro-oncology. One of the key aspects in this regard is the development of objective and standardized criteria that allow for accurate response assessment in clinical trials and prevent the misclassification of responders and non-responders. Such misclassification may lead to premature discontinuation of an actually effective agent, thereby withholding a potentially active treatment from the patient. Conversely, patients may be inappropriately continued on an inactive treatment. Moreover, such misclassification may confound the data obtained in such studies and may lead to false conclusions with regards to the efficacy of the investigated drug. Therefore, reliable response assessment criteria are necessary that not only accurately capture radiographic changes but also account for treatment-related changes and incorporate the assessment of clinical status and quality of life (QoL). The Response Assessment in Neuro-Oncology (RANO) working group is an international collaboration of neuro-oncologists, medical oncologists, radiation oncologists, neurosurgeons, neuroradiologists, and regulatory groups (among others) commissioned to develop objective and tumor-specific response criteria for various tumor subtypes. This article reviews the currently available response criteria for high-grade glioma, low-grade glioma, and brain metastases and discusses some of the barriers to accurate assessment of treatment response in neuro-oncology.
Brainstem gliomas in adults are a rare and heterogeneous group of brain tumors that vary with regard to underlying pathology, radiographic appearance, clinical course and prognosis. Diffuse intrinsic pontine gliomas represent the most common subtype. Although still considered aggressive and most often lethal, these brain tumors are associated with a more insidious clinical course and more favorable prognosis compared to the highly aggressive form in children. Treatment options for patients with brainstem gliomas still are limited and insufficiently studied. A better understanding of the pathobiology of these tumors will be crucial for the development of more specific and effective therapies. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2799-2809. © 2016 American Cancer Society.
When normal subjects fix their eyes upon a stationary target, their gaze is not perfectly still, due to small movements that prevent visual fading. Visual loss is known to cause greater instability of gaze, but reported comparisons with normal subjects using reliable measurement techniques are few. We measured binocular gaze using the magnetic search coil technique during attempted fixation (monocular or binocular viewing) of 4 individuals with childhood-onset of monocular visual loss, 2 individuals with late-onset monocular visual loss due to age-related macular degeneration, 2 individuals with bilateral visual loss, and 20 healthy control subjects. We also measured saccades to visual or somatosensory cues. We tested the hypothesis that gaze instability following visual impairment is caused by loss of inputs that normally optimize the performance of the neural network (integrator), which ensures both monocular and conjugate gaze stability. During binocular viewing, patients with early-onset monocular loss of vision showed greater instability of vertical gaze in the eye with visual loss and, to a lesser extent, in the normal eye, compared with control subjects. These vertical eye drifts were much more disjunctive than upward saccades. In individuals with late monocular visual loss, gaze stability was more similar to control subjects. Bilateral visual loss caused eye drifts that were larger than following monocular visual loss or in control subjects. Accurate saccades could be made to somatosensory cues by an individual with acquired blindness, but voluntary saccades were absent in an individual with congenital blindness. We conclude that the neural gaze-stabilizing network, which contains neurons with both binocular and monocular discharge preferences, is under adaptive visual control. Whereas monocular visual loss causes disjunctive gaze instability, binocular blindness causes both disjunctive and conjugate gaze instability (drifts and nystagmus). Inputs that bypass this neural network, such as projections to motoneurons for upward saccades, remain conjugate.
The elaboration of novel pathogenic aspects of multiple sclerosis (MS) requires the analysis of well-defined stages of lesion development. However, specimens of certain stages and lesion types are either present in small brain biopsies, insufficient in size for further molecular studies or available as formalin-fixed and paraffin-embedded (FFPE) material only. Therefore, application of current molecular biology techniques to FFPE tissue is warranted. We compared FFPE and frozen tissue by using quantitative polymerase chain reaction and report: (1) FFPE material is highly heterogeneous regarding the utility for transcript profiling of mRNAs; well-preserved FFPE samples had about a 100-fold reduced sensitivity compared with frozen tissue, but gave similar results for genes of sufficient abundance; (2) FFPE samples not suitable for mRNA analysis are still highly valuable for miRNA quantification; (3) the length of tissue fixation greatly affects utility for mRNA but not for miRNA analysis; (4) FFPE samples can be processed via a hot water bath for dissection of defined lesion areas; and (5) in situ hybridization for proteolipid protein (PLP) helps to identify samples not suitable for mRNA amplification. In summary, we present a detailed protocol how to use autoptic FFPE tissue for transcript profiling in dissected tissue areas.
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