Regional and Cellular Mapping of Sortilin Immunoreactivity in Adult Human Brain

Sortilin is a member of the vacuolar protein sorting 10 protein (VPS10P) domain receptor family, which carries out signal transduction and protein transport in cells. Sortilin serves as the third, G-protein uncoupled, receptor of neurotensin that can modulate various brain functions. More recent data indicate an involvement of sortilin in mood disorders, dementia and Alzheimer-type neuropathology. However, data regarding the normal pattern of regional and cellular expression of sortilin in the human brain are not available to date. Using postmortem adult human brains free of neuropathology, the current study determined sortilin immunoreactivity (IR) across the entire brain. Sortilin IR was broadly present in the cerebrum and subcortical structures, localizing to neurons in the somatodendritic compartment, but not to glial cells. In the cerebrum, sortilin IR exhibited differential regional and laminar patterns, with pyramidal, multipolar and polymorphic neurons in cortical layers II–VI, hippocampal formation and amygdaloid complex more distinctly labeled relative to GABAergic interneurons. In the striatum and thalamus, numerous small-to-medium sized neurons showed light IR, with a small group of large sized neurons heavily labeled. In the midbrain and brainstem, sortilin IR was distinct in neurons at the relay centers of descending and ascending neuroanatomical pathways. Dopaminergic neurons in the substantia nigra, cholinergic neurons in the basal nuclei of Meynert and noradrenergic neurons in the locus coeruleus co-expressed strong sortilin IR in double immunofluorescence. In comparison, sortilin IR was weak in the olfactory bulb and cerebellar cortex, with the mitral and Purkinje cells barely visualized. A quantitative analysis was carried out in the lateral, basolateral, and basomedial nuclei of the amygdaloid complex, as well as cortical layers II–VI, which established a positive correlation between the somal size and the intensity of sortilin IR among labeled neurons. Together, the present study demonstrates a predominantly neuronal expression of sortilin in the human brain with substantial regional and cell-type variability. The enriched expression of sortilin in pyramidal, dopaminergic, noradrenergic and cholinergic neurons suggests that this protein may be particularly required for signal transduction, protein trafficking and metabolic homeostasis in populations of relatively large-sized projective neurons

Bioelectromagnetics research within an Australian context: the Australian centre for electromagnetic bioeffects research (ACEBR)

Mobile phone subscriptions continue to increase across the world, with the electromagnetic fields (EMF) emitted by these devices, as well as by related technologies such as Wi-Fi and smart meters, now ubiquitous. This increase in use and consequent exposure to mobile communication (MC)-related EMF has led to concern about possible health effects that could arise from this exposure. Although much research has been conducted since the introduction of these technologies, uncertainty about the impact on health remains. The Australian Centre for Electromagnetic Bioeffects Research (ACEBR) is a National Health and Medical Research Council Centre of Research Excellence that is undertaking research addressing the most important aspects of the MC-EMF health debate, with a strong focus on mechanisms, neurodegenerative diseases, cancer, and exposure dosimetry. This research takes as its starting point the current scientific status quo, but also addresses the adequacy of the evidence for the status quo. Risk communication research complements the above, and aims to ensure that whatever is found, it is communicated effectively and appropriately. This paper provides a summary of this ACEBR research (both completed and ongoing), and discusses the rationale for conducting it in light of the prevailing science.Sarah P. Loughran ... Jim Manavis ... Robert Vink ... et al

APOMAB®, a La-Specific Monoclonal Antibody, Detects the Apoptotic Tumor Response to Life-Prolonging and DNA-Damaging Chemotherapy

Background: Antineoplastic therapy may impair the survival of malignant cells to produce cell death. Consequently, direct measurement of tumor cell death in vivo is a highly desirable component of therapy response monitoring. We have previously shown that APOMAB® representing the DAB4 clone of a La/SSB-specific murine monoclonal autoantibody is a malignant cell-death ligand, which accumulates preferentially in tumors in an antigen-specific and dose-dependent manner after DNA-damaging chemotherapy. Here, we aim to image tumor uptake of APOMAB® (DAB4) and to define its biological correlates. Methodology/Principal Findings: Brisk tumor cell apoptosis is induced in the syngeneic EL4 lymphoma model after treatment of tumor-bearing mice with DNA-damaging cyclophosphamide/etoposide chemotherapy. Tumor and normal organ accumulation of Indium 111 (111In)-labeled La-specific DAB4 mAb as whole IgG or IgG fragments was quantified by whole-body static imaging and organ assay in tumor-bearing mice. Immunohistochemical measurements of tumor caspase-3 activation and PARP-1 cleavage, which are indicators of early and late apoptosis, respectively, were correlated with tumor accumulation of DAB4. Increased tumor accumulation of DAB4 was associated directly with both the extent of chemotherapy-induced tumor cell death and DAB4 binding per dead tumor cell. Tumor DAB4 accumulation correlated with cumulative caspase-3 activation and PARP-1 cleavage as tumor biomarkers of apoptosis and was directly related to the extended median survival time of tumor-bearing mice. Conclusions/Significance: Radiolabeled La-specific monoclonal antibody, DAB4, detected dead tumor cells after chemotherapy, rather than chemosensitive normal tissues of gut and bone marrow. DAB4 identified late apoptotic tumor cells in vivo. Hence, radiolabeled DAB4 may usefully image responses to human carcinoma therapy because DAB4 would capture the protracted cell death of carcinoma. We believe that the ability of radiolabeled DAB4 to rapidly assess the apoptotic tumor response and, consequently, to potentially predict extended survival justifies its future clinical development as a radioimmunoscintigraphic agent. This article is part I of a two-part series providing proof-of-concept for the the diagnostic and therapeutic use of a La-specific monoclonal antibody, the DAB4 clone of which is represented by the registered trademark, APOMAB®.Fares Al-Ejeh, Jocelyn M. Darby, Chris Tsopelas, Douglas Smyth, Jim Manavis and Michael P. Brow

Nutlin-3a efficacy in sarcoma predicted by transcriptomic and epigenetic profiling

Nutlin-3a is a small-molecule antagonist of p53/MDM2 that is being explored as a treatment for sarcoma. In this study, we examined the molecular mechanisms underlying the sensitivity of sarcomas to Nutlin-3a. In an ex vivo tissue explant system, we found that TP53 pathway alterations (TP53 status, MDM2/MDM4 genomic amplification/mRNA overexpression, MDM2 SNP309, and TP53 SNP72) did not confer apoptotic or cytostatic responses in sarcoma tissue biopsies (n = 24). Unexpectedly, MDM2 status did not predict Nutlin-3a sensitivity. RNA sequencing revealed that the global transcriptomic profiles of these sarcomas provided a more robust prediction of apoptotic responses to Nutlin-3a. Expression profiling revealed a subset of TP53 target genes that were transactivated specifically in sarcomas that were highly sensitive to Nutlin-3a. Of these target genes, the GADD45A promoter region was shown to be hypermethylated in 82% of wild-type TP53 sarcomas that did not respond to Nutlin-3a, thereby providing mechanistic insight into the innate ability of sarcomas to resist apoptotic death following Nutlin-3a treatment. Collectively, our findings argue that the existing benchmark biomarker for MDM2 antagonist efficacy (MDM2 amplification) should not be used to predict outcome but rather global gene expression profiles and epigenetic status of sarcomas dictate their sensitivity to p53/MDM2 antagonists.Kathleen I. Pishas, Susan J. Neuhaus, Mark T. Clayer, Andreas W. Schreiber, David M. Lawrence, Michelle Perugini, Robert J. Whitfield, Gelareh Farshid, Jim Manavis, Steve Chryssidis, Bronwen J. Mayo, Rebecca C. Haycox, Kristen Ho, Michael P. Brown, Richard J. D'Andrea, Andreas Evdokiou, David M. Thomas, Jayesh Desai, David F. Callen and Paul M. Neilse

A biomechanical model of traumatic contusional injury produced by controlled cerebrocortical indentation in sheep

International audienceA biomechanical model of traumatic contusional injury was used to map axonal damage and neuronal reaction proximal and distal from the contusion. The model uses a precisely controlled and characterised dynamic indentation of the cerebral cortex of anaesthetised sheep. The indentation (16.15-16.50 mm deep; contact speed 1.2-1.24 m/s) is made through a 20 mm craniotomy in the frontal bone. The brain is then perfused-fixed after 6 hours and sectioned at 5 mm intervals. Immunohistochemistry was used to detect axonal injury and neuronal reaction. Quantitation of injury was by an automatic counting algorithm applied to micrographs of each entire section. These maps were cross-checked with manual counts. The injury was characterised by well-defined zones radiating from the impact point; these were a region of haemorrhagic and necrotic tissue, subadjacent penumbra of axonal injury, and distal multi-focal and diffuse areas of neuronal positivity. The model includes precise characterisation of the contact load and the pattern of injury. This will allow future finite element modelling to be used to explore quantitative relationships between several forms of neural damage and the dynamics of the tissue deformation in a finite element model of the insult

Cytotoxic T Cells Are the Predominant Players Providing Cross-Protective Immunity Induced by γ-Irradiated Influenza A Viruses ▿

We previously demonstrated that a single dose of nonadjuvanted intranasal γ-irradiated influenza A virus can provide robust protection in mice against both homologous and heterosubtypic challenges, including challenge with an H5N1 avian virus strain. We investigated the mechanism behind the observed cross-protection to define which arms of the adaptive immune response are involved in mediating this protection. Studies with gene knockout mice showed the cross-protective immunity to be mediated mainly by T cells and to be dependent on the cytolytic effector molecule perforin. Adoptive transfer of memory T cells from immunized mice, but not of memory B cells, protected naïve recipients against lethal heterosubtypic influenza virus challenge. Furthermore, γ-irradiated influenza viruses induced cross-reactive Tc-cell responses but not cross-neutralizing or cross-protective antibodies. In addition, histological analysis showed reduced lung inflammation in vaccinated mice compared to that in unvaccinated controls following heterosubtypic challenge. This reduced inflammation was associated with enhanced early recruitment of T cells, both CD4+ and CD8+, and with early influenza virus-specific cytotoxic T-cell responses. Therefore, cross-protective immunity induced by vaccination with γ-irradiated influenza A virus is mediated mainly by Tc-cell responses

Neutralizing monoclonal antibodies to different clades of Influenza A H5N1 viruses

Four IgG1� monoclonal antibodies (mAbs) against Influenza A/Chicken/Vietnam/8/2004 (H5N1) virus are described. Three of these showed neutralizing activities against H5N1 strains from clades 1, 2 and 3 using a retroviral pseudotype or live virus microneutralization assay. In the pseudotype assay, the IC90 neutralizing titre range was >1600–51,200, and with the microneutralization was 80≥10,240. MAb 1C1 showed strong neutralizing activities in both assays. All four mAbs reacted specifically to the immunogen by immunohistochemical staining and to A/Hong Kong/483/1997 (H5N1) and A/Thailand/1(KAN-1)/2004 (H5N1)-infected MDCK cells by immunofluorescence. ELISA titrations of the mAbs showed specificity for H5N1 haemagglutinin (HA) and no cross-reactivity to 15 other Influenza A subtypes. Only mAbs 1C1 and the non-neutralizing 1F7 reacted with HA1, the cleaved subunit of HA, by Western blot. These results suggest that the mAbs recognize distinct or overlapping epitopes and will be useful reagents for construction of specific rapid point-of-care assays or for therapeutic use