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Dropped head syndrome (DHS) has been rarely observed in amyotrophic lateral sclerosis (ALS), and the neuropathological findings of this condition have almost never been described. The identification of transactivation response DNA‐binding protein 43 kDa (TDP‐43), which binds to RNA/DNA has provided a new method for studying ALS and frontotemporal lobar degeneration (FTLD). Post‐mortem examination of an adult sudden death case of a 71‐year‐old patient who complained of DHS exhibited severe loss of anterior motor neurons in the cervical cord (C4‐6). Loss of nerve fibers of the anterior roots was striking compared with posterior roots, together with marked neurogenic atrophy of posterior muscles semispinalis cervicis. Bunina bodies were found in large neurons of Betz giant cells, but not in the motor neurons of spinal cords, or neurons of bulbar regions. Phosphorylated TDP‐43 (p‐TDP‐43)‐positive structures were detected in the residual neurons of the cervical, thoracic and lumber cords, hypoglossal nucleus, cerebellar dentate nucleus and parahippocampal cortex, together with ubiquitin‐positive inclusions. Phosphorylated Tau positive structures in neuronal cytoplasm were found in the amygdala, entorhinal cortex and parahippocampal cortex, some of which co‐expressed p‐TDP‐43. The medial zone of cervical cords may be the first onset site, and that is the cause of head drop in the early stage of ALS. In spite of detailed examination, the direct cause of sudden death was not verified. This autopsy report revealed the relation of DHS which is a rare clinical manifestation of ALS, and neuropathological findings. [ABSTRACT FROM AUTHOR]

A 24 year‐old female presented with a mass lesion in the right temporal lobe. This case was difficult to diagnose using histological and immunological methods and therefore molecular analyses were applied to provide a definitive diagnosis. The tumor was well‐demarcated, partially cystic, and irregularly‐enhanced on gadolinium‐enhanced T1‐weighted magnetic resonance images. Pathologically, a large part of the tumor consisted of cells with fine cytoplasmic processes on a myxoid and mucinous background. Cells formed a microcystic structure around the mucinous tissue. Numerous eosinophilic granular bodies, but not Rosenthal fibers, were present. The solid and compact regions of the tumor were composed of fasciculation of dense fibrous glial tissues and occasional multinucleated giant cells. Tumor cells and their fragmented cytoplasmic processes were positively stained with GFAP, while eosinophilic granular bodies were both positive and negative. Xanthomatous changes were not detected and the reticulin fibers were restricted to vascular tissues. The MIB1 index was scored as approximately 10%. In molecular analyses of BRAF, the KIAA1549‐BRAF (K16‐B9) fusion gene was detected in all tumor regions, whereas BRAF V600E mutation was not detected by either conventional Sanger sequencing or the Eprobe‐PCR method. Based on the results of the molecular analyses, this case was diagnosed as pilocytic astrocytoma. [ABSTRACT FROM AUTHOR]

Enteroviruses (EVs) are associated with a broad spectrum of clinical presentation, including aseptic meningitis (AM), encephalitis, hand, foot and mouth disease, acute flaccid paralysis, and acute flaccid myelitis. Epidemics occur sporadically and are associated with increased cases of AM in children. The present study describes the seroepidemiological analysis of circulating EVs in Ireland from 2005 to 2014 and phylogenetic characterization of echovirus 30 (E-30), enterovirus A71 (EV-A71), and enterovirus D68 (EV-D68). EV VP1 genotyping was applied to viral isolates and clinical samples, including cerebrospinal fluid (CSF), and those isolates that remained untypeable by neutralising anti-sera. An increase in AM cases from 2010 to 2014 was associated with an E-30 genogroup variant VII and sequences clustered phylogenetically with those detected in AM outbreaks in France and Italy. EV-D68 viral RNA was not detected in CSF samples and no neurological involvement was reported. Three EV-A71 positive CSF samples were identified in patients presenting with AM. A phylogenetic analysis of respiratory-associated EV-D68 and EV-A71 cases in circulation was performed to determine baseline epidemiological data. EV-D68 segregated with clades B and B(1) and EV-A71 clustered as subgenogroup C2. The EV VP1 genotyping method was more sensitive than neutralising anti-sera methods by virus culture and importantly demonstrated concordance between EV genotypes in faecal and CSF samples which should facilitate EV screening by less invasive sampling approaches in AM presentations. [ABSTRACT FROM AUTHOR]

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins. [ABSTRACT FROM AUTHOR]

Studies have identified solid organ transplant recipients who remain asymptomatic despite maintaining CHL. Factors which determine the CHL state remain poorly understood but are likely to involve immunological control of the viral infection. We monitored expression of PD-1, a marker of T-cell exhaustion and viral persistence, on CD8 T cells in patients who resolved EBV infection as determined by undetectable EBV DNA ( REI) and CHL patients. PD-1 expression on CD8 T cells was increased in the first year post-transplant irrespective of EBV outcome, and most CD8 T cells continued to express PD-1 for up to three yr post-transplant. Although all patient groups showed similar frequencies of EBV-specific CD8+ T cells, PD-1 expression on these cells increased in the post-transplant groups compared with the pretransplant patients. Functional studies of EBV-specific CD8+ T cells stimulated with BZLF or LMP2 peptide pools revealed monofunctional IFN-γ responses. Our results indicate that PD-1 expression on CD8 T cells post-transplant may result from factors other than antigenic stimulation. [ABSTRACT FROM AUTHOR]

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome. [ABSTRACT FROM AUTHOR]

The molecular characterization of measles virus (MeV) is a valuable epidemiological tool to monitor virus transmission and to discriminate between imported and endemic infection. There has been significant immigration into Ireland in recent years and many individuals originate from regions of high measles incidence. Ireland has had a number of outbreaks of MeV which appear attributable to sub-optimal vaccine uptake and possibly imported strains as new genotypes have been identified in recent years. To ascertain any significant changes in circulating measles genotypes we investigated 65 confirmed measles cases between the years 2002 and 2007. The laboratory diagnosis of measles was confirmed by detection of measles-specific IgM in oral fluid in conjunction with a real-time polymerase chain reaction assay targeting the MeV hemagglutinin gene. Phylogenetic analysis based on the 3′ hypervariable region of the nucleoprotein gene was performed and three genotypes, all within measles clade D, were found to be circulating during this time period. In 2002 and 2003, genotype D8 (n = 2) was observed whereas genotype D7 was dominant in 2003 (n = 31). A distinct change in the circulating MeV genotype and increased genetic diversity was observed between 2004 and 2007. All cases were within genotype D4 (n = 32) but were phylogenetically distinct from each other. These data provide important epidemiologic baseline information on MeV in Ireland and facilitates detailed examination of measles transmission. J. Med. Virol. 81:125-129, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real-time RT-PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real-time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub-clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT-PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44-8.15). Statistical analysis of quantitative RT-PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates ( P < 0.03). J. Med. Virol. 80:510-516, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein (Agno) has been shown to bind to HP1aand to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1afrom LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus. [ABSTRACT FROM AUTHOR]

HTLV-2 is a deltaretrovirus, possibly associated with neurological manifestations. Initially believed to be restricted to CD8+ lymphocytes, the virus has been shown to infect also CD4+ T-cells and B cells. Here we show for the first time that HTLV-2 also infects NK cells in vivo. HTLV-2-infected subjects were enrolled. PBMCs isolated by Ficoll density gradient were submitted to sequential isolation using MicroBeads (Miltenyi Biotech), in the following order: CD4+, CD56+, CD8+ and CD19+. For flow cytometry sorting in a BD FACSAria, cells were stained with a combination of anti-human surface markes (all from BD Biosciences). DNA from the subsets and from total PBMCs was extracted. Proviral load (PVL) measurement was done by a multiplex real-time PCR assay, targeting a fragment of HTLV-2 Tax gene and a fragment of human albumin gene. 6 samples had undetectable PVL (bellow 1 Tax copy/100 PBMCs). A comparison CD8+ T cells showed the highest proviral burden, followed by CD56+ cells, CD19+ cells and CD4+ cells (mean PVLs= 17.84, 11.7, 7.27 and 0.47 respectively). To further exclude that the PVL detected in the NK subset was due to contaminants, total PBMCs from 2 samples were stained for FACS sorting. Both the 'classic' NK cells and NK cells expressing CD8+ were found to be infected. This study provides for the first time evidence that NK cells are naturally infected by HTLV-2, and constitute a major reservoir of infection. Potentially, infected NK cells might be functionally impaired, resulting in viral escape from the innate immune surveillance. A clearer assessment of the in vivo spectra of the infection is essential for understanding virus - host interactions and the mechanisms of virus persistence. [ABSTRACT FROM AUTHOR]