Activation of NR1a/NR2B receptors by soluble factors from APP-stimulated monocyte-derived macrophages: implications for the pathogenesis of Alzheimer's disease

Amyloid-beta peptide (Abeta), the major component of amyloid plaques, can activate brain mononuclear phagocytes (MP; macrophages and microglia), leading to their secretion of neurotoxins. Recent studies strongly suggest that MP-mediated neurotoxicity plays an important role in the pathogenesis of Alzheimer's disease (AD). To further explore this notion, human monocyte-derived macrophages (MDM) were stimulated with naturally secreted alpha-processing soluble amyloid precursor protein/p3 (alphaAPPs/p3) or beta-processing APP/Abeta (betaAPPs/Abeta). MDM conditioned media (MCM) was recovered and tested for its ability to activate recombinant N-methyl-d-aspartate (NMDA) receptor subtype NR1a/NR2B expressed in Xenopus oocytes. Pressure ejection of alphaAPPs/p3- and betaAPPs/Abeta-stimulated MCM produced inward currents of 59.5 +/- 8.9 nA (mean +/- S.E.M., n = 31) and 111.1 +/- 21.0 nA (n = 42) in NR1a/NR2B-expressing oocytes, respectively. The MCM-induced currents were concentration dependent and blocked by 50 microM of the NMDA receptor antagonist 2-amino-5-phosphnovalerate, but not by a non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). The alphaAPPs/p3- and betaAPPs/Abeta-stimulated MCM placed in non-injected oocytes failed to generate inward current. These results demonstrate that APPs/Abeta-stimulated MCM directly activate NMDA receptor subtypes relevant in the pathogenesis of AD.     

Utilization of the 2017 diagnostic criteria for hEDS by the Toronto GoodHope Ehlers–Danlos syndrome clinic: A retrospective review

The new 2017 diagnostic criteria for hypermobile Ehlers–Danlos Syndrome (hEDS) provide a framework for diagnosing hEDS but are more stringent than the previous Villefranche criteria. Our clinical experience at the GoodHope EDS clinic was that the 2017 criteria left many highly symptomatic patients without a diagnosis of hEDS. We conducted a retrospective cohort study to confirm our clinic experience and assess the accuracy of the 2017 diagnostic criteria for hEDS in patients who had a previous hEDS diagnosis based on the Villefranche criteria. Our study found that 15% (n = 20 of 131) of patients with a prior diagnosis of hEDS met the 2017 diagnostic criteria, and many of the traits used to distinguish hEDS were not significantly more frequent in patients who met 2017 criteria versus those who did not. In both groups objective systemic manifestations were found less frequently than subjective systemic manifestations. Beighton score (BS) as assessed by primary care practitioner was found to be higher than assessment by EDS practitioner in 81% (n = 74 of 91) of cases. Generalized joint hypermobility was confirmed in only 46% (n = 51 of 111) of patients who had a previous diagnosis of hEDS. Higher BS did not correlate with increased number of systemic manifestations in our cohort. Common comorbidities of hEDS were found with similar frequency in those who met 2017 criteria and those who did not. Based on our cohort, the 2017 hEDS diagnostic criteria require refinement to improve its diagnostic accuracy.     

Antigenic characterisation of influenza B virus with a new microneutralisation assay: comparison to haemagglutination and sequence analysis

Although the haemagglutination inhibition assay is considered the "gold standard" for antigenic characterisation of influenza viruses, some limitations of this technique are well known. A new microneutralisation assay, as a tool for antigenic characterisation of influenza B viruses, has been standardised and its performance evaluated in comparison with the haemagglutination inhibition test in the light of molecular characterisation of the haemagglutinin. Twelve B viruses belonging to the two lineages and the four sub-lineages discriminated by phylogenetic analysis of HA were tested. The microneutralisation assay clearly distinguishes viruses belonging to different lineages and, in addition, discriminates strains belonging to different sub-lineages that are poorly or not discriminated using the haemagglutination inhibition test. This new microneutralisation assay could provide a useful tool for antigenic characterisation of circulating influenza viruses and contribute, together with the haemagglutination inhibition test and sequence analysis of the haemagglutinin and neuraminidase, in the choice of the strain for use in vaccine composition.     

Amphiphysin I phosphorylation on residue threonine 260 in a pentylenetetrazole-induced seizure model

A method to evaluate kinase inhibitor action was reported [L. Morgan, S.J. Neame, H. Child, R. Chung, B. Shah, L. Barden, J.M. Staddon, T.R. Patel, Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system, Neurosci. Lett. 395 (2006) 143–148]. In this, acute administration of the GABA antagonist pentylenetetrazole triggers seizures through glutamate-dependent pathways. Under such conditions, activation of the c-Jun N-terminal kinase (JNK) pathway was detected in hippocampal extracts. Phosphorylation of the upstream JNK kinase MKK4 was also revealed through use of a phospho-MKK4-specific antibody. Here, this antibody is shown to also react with a protein of ∼125 kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the ∼125 kDa protein as it may provide novel insight into signalling, neuronal activity and seizures. Using chromatographic methods and mass spectrometry, the protein was identified as amphiphysin I. This was confirmed by 2D gel analysis and immunoblot with amphiphysin I-specific antibodies. Although the phospho-MKK4 antibody was raised against an MKK4-specific peptide, partial sequence homology between this sequence and a region of amphiphysin was discerned. New antibodies raised against the phospho-threonine 260-amphiphysin-specific sequence detected increased phosphorylation in response to pentylenetetrazole treatment. This particular phosphorylation site does not seem to have been described before, possibly reflecting a novel regulatory aspect of amphiphysin biology. As amphiphysin is involved in the regulation of endocytosis, phosphorylation at this site may play a role in the regulated re-uptake of synaptic vesicles after neurotransmitter release.     

Radial gradient-based segmentation of mammographic microcalcifications: observer evaluation and effect on CAD performance

Precise segmentation of microcalcifications is essential in the development of accurate mammographic computer-aided diagnosis (CAD) schemes. We have designed a radial gradient-based segmentation method for microcalcifications, and compared it to both the region-growing segmentation method currently used in our CAD scheme and to the watershed segmentation method. Two observer studies were conducted to subjectively evaluate the proposed segmentation method. The first study (A) required observers to rate the segmentation accuracy on a 100-point scale. The second observer evaluation (B) was a preference study in which observers selected their preferred method from three displayed segmentation methods. In study A, the observers gave an average accuracy rating of 88 for the radial gradient-based and 50 for the region-growing segmentation method. In study B, the two observers selected the proposed method 56% and 62% of the time. We also investigated the effect of the proposed segmentation method on the performance of computerized classification scheme in differentiating malignant from benign clustered microcalcifications. The performances of the classification scheme using a linear discriminant analysis (LDA) or a Bayesian artificial neural network classifier both showed statistically significant improvements when using the proposed segmentation method. The areas under the receiver-operating characteristic curves for case-based performance when using the LDA classifier were 0.86 with the proposed segmentation method, 0.80 with the region-growing method, and 0.83 with the watershed method.