HPV testing on self collected cervicovaginal lavage specimens as screening method for women who do not attend cervical screening: cohort study

Objective To determine whether offering self sampling of cervicovaginal material for high risk human papillomavirus (HPV) testing is an effective screening method for women who do not attend regular cervical screening programmes

Spatial concordance of DNA methylation classification in diffuse glioma.

BACKGROUND: Intratumoral heterogeneity is a hallmark of diffuse gliomas. DNA methylation profiling is an emerging approach in the clinical classification of brain tumors. The goal of this study is to investigate the effects of intratumoral heterogeneity on classification confidence. METHODS: We used neuronavigation to acquire 133 image-guided and spatially separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma (7 IDH-wildtype and 2 IDH-mutant glioblastoma, 6 diffuse astrocytoma, IDH-mutant and 1 oligodendroglioma, IDH-mutant and 1p19q codeleted), which we characterized using DNA methylation arrays. Samples were obtained from regions with and without abnormalities on contrast-enhanced T1-weighted and fluid-attenuated inversion recovery MRI. Methylation profiles were analyzed to devise a 3-dimensional reconstruction of (epi)genetic heterogeneity. Tumor purity was assessed from clonal methylation sites. RESULTS: Molecular aberrations indicated that tumor was found outside imaging abnormalities, underlining the infiltrative nature of this tumor and the limitations of current routine imaging modalities. We demonstrate that tumor purity is highly variable between samples and explains a substantial part of apparent epigenetic spatial heterogeneity. We observed that DNA methylation subtypes are often, but not always, conserved in space taking tumor purity and prediction accuracy into account. CONCLUSION: Our results underscore the infiltrative nature of diffuse gliomas and suggest that DNA methylation subtypes are relatively concordant in this tumor type, although some heterogeneity exists

Coexpression of vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex

We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes. An intense, punctate VGluT1-staining occurred everywhere in the entorhinal cortex. Our computer program resolved these punctae as small 3D objects. Also VGluT2 showed a punctate immunostaining pattern, yet with half the number of 3D objects per tissue volume compared with VGluT1, and with statistically significantly larger 3D objects. Both VGluTs were distributed homogeneously across cortical layers, with in MEA VGluT1 slightly more densely distributed than in LEA. The distribution pattern and the size distribution of GAD 3D objects resembled that of VGluT2. CR-immunopositive fibers were abundant in all cortical layers. In double-stained sections we noted ample colocalization of CR and VGluT2, whereas coexpression of CR and VGluT1 was nearly absent. Also in triple-staining experiments (VGluT2, GAD and CR combined) we noted coexpression of VGluT2 and CR and, in addition, frequent coexpression of GAD and CR. Modest colocalization occurred of VGluT2 and GAD, and incidental colocalization of all three markers. We conclude that the CR-containing axon terminals in the entorhinal cortex belong to at least two subpopulations of CR-neurons: a glutamatergic excitatory and a GABAergic inhibitory

Cholinergic imbalance in the multiple sclerosis hippocampus

Hippocampal pathology was shown to be extensive in multiple sclerosis (MS) and is associated with memory impairment. In this post-mortem study, we investigated hippocampal tissue from MS and Alzheimer's disease (AD) patients and compared these to non-neurological controls. By means of biochemical assessment, (immuno)histochemistry and western blot analyses, we detected substantial alterations in the cholinergic neurotransmitter system in the MS hippocampus, which were different from those in AD hippocampus. In MS hippocampus, activity and protein expression of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was decreased, while the activity and protein expression of acetylcholinesterase (AChE), the acetylcholine degrading enzyme, was found to be unaltered. In contrast, in AD hippocampus both ChAT and AChE enzyme activity and protein expression was decreased. Our findings reveal an MS-specific cholinergic imbalance in the hippocampus, which may be instrumental in terms of future treatment options for memory problems in this diseas

Computer-assisted estimation in the CNS of 3D multimarker 'overlap' or 'touch' at the level of individual nerve endings: a confocal laser scanning microscope application

Presynaptic boutons and associated postsynaptic structures in the CNS express markers that are highly synapse type-specific. In multilabel immunofluorescence imaging, coexpression of such markers appears as overlap of signals in the same structures whereas closely related yet segregated markers, e.g., located pre-and postsynaptically, translate into signals that touch. 'Overlap' and 'touch' occur in three dimensions (3D). The instrument of choice to study overlap vs. touch of small objects in tissue volumes is the confocal laser scanning microscope (CSLM). To quantify overlap and touch we used two paradigms. Overlap was studied in rat brain sections triple-immunostained with antibodies against markers predominantly located presynaptically: glutamic acid decarboxylase, vesicular glutamate transporter 2, and calretinin. Touch was studied in rat temporal cortex where afferent, tracer-labeled entorhinohippocampal fibers in the subiculum were imaged together with possible postsynaptic target neurons immunostained with an antibody against the calcium binding protein, parvalbumin. Z-series of CLSM images were obtained in multiple channels. After post-acquisition deconvolution we further processed the images via software written in the C/C++ SCIL Image computer programming environment. The software receives parameters via scripts, after which it first identifies 3D objects through establishing isodensity envelopes around pixels representing small biologic structures (in our case: boutons) and then compares associated Z-series in which it determines whether there is overlap or touch between recognized 3D objects. Finally, graphic and numeric output is produced. With this script-commanded software we feel equipped to accurately and objectively quantify overlap and touch

Astrocytic Abeta1-42 uptake is determined by Abeta-aggregation state and the presence of amyloid-associated proteins

Intracerebral accumulation of amyloid-beta (Abeta) leading to Abeta plaque formation, is the main hallmark of Alzheimer's disease and might be caused by defective Abeta-clearance. We previously found primary human astrocytes and microglia able to bind and ingest Abeta1-42 in vitro, which appeared to be limited by Abeta1-42 fibril formation. We now confirm that astrocytic Abeta-uptake depends on size and/or composition of Abeta-aggregates as astrocytes preferably take up oligomeric Abeta over fibrillar Abeta. Upon exposure to either fluorescence-labelled Abeta1-42 oligomers (Abeta(oligo)) or fibrils (Abeta(fib)), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. Abeta-internalization was verified using confocal microscopy and live-cell imaging. Neither uptake of Abeta(oligo) nor Abeta(fib), triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin-6 and monocyte-chemoattractant protein-1 release. Amyloid-associated proteins, including alpha1-antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence Abeta-aggregation. Here, astrocytic uptake of Abeta(fib) increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of Abeta(oligo)-positive astrocytes, whereas SAP/C1q slightly reduced Abeta(oligo) uptake. Thus, amyloid-associated proteins, especially ApoJ and ApoE, can alter Abeta-uptake in vitro and hence may influence Abeta clearance and plaque formation in vivo. (c) 2010 Wiley-Liss, In

Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex

Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition. In primary visual cortex (area V1), the density and subunit composition of AMPARs differ among cortical layers and among cell types. The AMPARs expressed by the different types of inhibitory interneurons, which are crucial for network function, have not yet been characterized systematically. We investigated the distribution of AMPAR subunits in macaque V1 for three distinct subpopulations of inhibitory interneurons: parvalbumin-immunoreactive (PV-IR) interneurons, calbindin-immunoreactive (CB-IR) interneurons, and calretinin-immunoreactive (CR-IR) interneurons. We found that PV-IR cells, which have previously been identified as fast spiking, show high expression of the GluA2 and GluA3 subunits. In contrast, CB-IR and CR-IR cells, which tend to be intermediate spiking, show high expression of the GluA1 and GluA4 subunits. Thus, our data demonstrate that the expression of AMPARs divides inhibitory interneurons in macaque V1 into two categories that are compatible with existing classification methods based on calcium-binding proteins and firing behavior. Moreover, our findings suggest new approaches to target the different inhibitory interneuron classes pharmacologically in viv

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing-immunofluorescence study in the rat

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs. Labeled amygdalostriatal fibers were seen when tracer had been injected into the magnocellular and parvicellular portions of the basal amygdaloid nucleus and the lateral amygdaloid nucleus (nuclei belonging to 'cortical type' amygdaloid nuclei). Intra-amygdaloidal projection fibers were mostly found after tracer injections in the central and medial amygdaloid nuclei ('striatal type' amygdaloid nuclei). Terminals of tracer-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 and to a lesser degree with VGluT2 or VGAT. Intra-amygdaloid labeled fibers showed colocalization mostly of VGluT1, followed by VGAT. VGluT2 co-occurred in a minority of intra-amygdaloid tracer-containing fiber terminals. We conclude from our observations that both amygdalostriatal and intra-amygdaloid projections, arising from, respectively, 'cortical type' and 'striatal type' amygdaloid nuclei contain strong glutamatergic and modest GABAergic components. The glutamatergic fibers express either VGluT1 or VGluT2. The absence in large numbers of tracer labeled fibers of expression of one of the selected VTs leads us to suspect that amygdalostriatal projection fibers may contain hitherto neglected neurotransmitters in these connections, e.g., aspartate