Browsing by Author "Lang, Dirk M"

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    Open Access
    Characterisation of innate fungal recognition in the lung
    (Public Library of Science, 2012) Faro-Trindade, Inês; Willment, Janet A; Kerrigan, Ann M; Redelinghuys, Pierre; Hadebe, Sabelo; Reid, Delyth M; Srinivasan, Naren; Wainwright, Helen; Lang, Dirk M; Steele, Chad
    The innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus , and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung.
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    The effect of voluntary exercise on adult hippocampal neurogenesis in maternally separated rats
    (2016) Hardcastle, Natasha Sema; Russell, Vivienne A; Lang, Dirk M; Marais, L
    Maternal separation (MS) has been shown to produce depression-like symptoms in male Sprague Dawley rats. The underlying mechanisms responsible for the development of these depression-like behaviors are unknown. However, a growing body of evidence suggests that a reduction in neurogenesis may be a key-mediating factor. Voluntary wheel running is a form of exercise that increases neurogenesis and decreases depression-like behaviour in rats. However, the exact molecular role of neurogenesis in exercise-induced antidepressant effects still remains unanswered. This requires new tools to explore the interact ion between exercise and neurogenesis in vivo. To this end, the novel mitotic-marker, 5-ethynyl-2'-deoxyuridine (EdU), and Ki-67, an endogenous marker of cell proliferation, was characterised in order to study neurogenesis in an MS rat model of depression. Furthermore, this study aimed to provide insight into the effect of voluntary exercise on cell genesis and survival. To characterise EdU labelling of cells in vivo, male Sprague Dawley rats (Characterisation rats n =13) were injected with 50 mg/kg EdU a s noted in the literature. The optimal time point to inject the EdU label to measure mitotic activity was found to be post-natal day (PND) 60. MS or non-maternal separation (NMS) was conducted from PND 2-14 on experimental rats (n=39). From PND 54 - 74, ex perimental rats were housed in cages with attached running wheels (R) or locked running wheels (NR). All experimental rats were injected with 50 mg/kg EdU on PND 60 and transcardially perfused on PND 74 using Phosphate Buffered Saline (PBS) followed by fre sh 4% paraformaldehyde. Whole brains were then removed from the skull and placed in 4% paraformaldehyde for three hours. The brains were transferred to a 30% sucrose solution, stored in sucrose for 3-5 days and thereafter mounted in optimal cutting mediu m (OCT) and sectioned using a cryostat. Brain sections of 40 μm from 6.96 to 5.52 mm anterior to the inter-aural line were taken as dorsal and 50 μm sections from 3.84 to 2.76 mm were analyzed as ventral. The marker, EdU was detected in rat brains using t he Click-iT EdU Alexa Fluor 488 detection kit. Three molecular marker combinations were used to detect different factors for both dorsal and ventral hippocampi: (1) EdU/GFAP/NeuN, to establish how many EdU labelled cells survive to become neurons or astroc ytes (2) EdU/DCX to determine how many EdU labelled cells that have survived for 14 days are immature neurons and (3) Ki-67/DCX to indicate how many mitotically active cells are immature neurons on PND 74. Brain sections were then scanned using a confocal microscope whereby EdU stained nuclei were manually counted and cell phenotypes identified. The molecular marker combination one and two revealed no differences between treatment groups in the number of EdU-labelled cells in the dorsal and ventral hippoca mpi. However, a significant correlation was found between EdU/GFAP positive cells and EdU/NeuN positive cells in the ventral hippocampus when all treatment groups were pooled (r = 0.82, n=18, p < 0.0001). The third molecular marker combination revealed sig nificant differences in neurogenesis between groups. The MS+R group had fewer dorsal hippocampal Ki-67/DCX cells relative to NMS+R and NMS+R had significantly higher Ki-67/DCX cell count relative to NMS+NR rats. In the ventral hippocampus MS+R rats had few er Ki-67/DCX cells compared to NMS+R rats. The link between neurons and astrocytes in the ventral hippocampi corresponds with reports that an increase in neurons is linked to the presence of astrocytes. However, it may also be due to unavoidable variation in the intensity of the stain. The third molecular marker combination (Ki-67/DCX) revealed the most significant finding of this study. It showed that voluntary wheel running significantly increased the number of Ki-67/DCX co-labelled neurons in the dorsal hippocampus of NMS+R rats relative to NMS+NR which is in agreement with the literature that suggests exercise increases neurogenesis. The literature also reports that stress decreases neurogenesis and interestin gly MS+R rats had a lower cell count than NMS+R rats. This may indicate an interaction between early life stress and exercise-induced neurogenesis. This finding further suggests that MS alters neurogenesis in adult life and attenuates the effect of exercis e on the ventral hippocampus.
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    Open Access
    The expression and functional analysis of neurite outgrowth inhibitors in the nervous system of Xenopus laevis
    (2007) Hsu, Nai-Jen; Lang, Dirk M
    Generally, the factors contributing to success or failure of axon regeneration lie in the intrinsic properties of the injured neurons, as well as the surrounding microenvironment of the transected axon. Mammalian neurons may lack the intrinsic ability to survive after trauma, or to re-express genes required for axonal regrowth. Moreover, several proteins inhibitory to neurite growth, such as Tenascin-R (TN-R) and Nogo-A, have been identified in mammals. These proteins are associated with oligodendrocytes and myelin and are considered major inhibitory components of the CNS environment.
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    The expression and functional role of Tenascin-R during axon regeneration in the adult goldfish, Carassius auratus
    (2006) McBride, Ruth; Lang, Dirk M
    Includes bibliographical references (leaves 126-144).
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    Localisation and expression pattern of the Nogo receptor and its ligand, Nogo-A in cells of the mammalian central nervous system
    (2006) Nyatia, Edward; Lang, Dirk M
    Axon regeneration failure in the adult mammalian central nervous system is partly due to inhibitory molecules associated with myelin. The Nogo receptor plays a role in this process through an extraordinary degree of cross reactivity with three structurally unrelated myelin-associated inhibitory ligands namely; Nogo-A, myelin associated glycoprotein and oligodendrocyte myelin glycoprotein. The major aim of the study was to investigate the expression pattern of Nogo receptor and one of its ligands, Nogo-A in the mammalian nervous system, and also investigate whether Nogo receptor is located in neuronal lipid rafts by linking it to flotillins, known lipid raft markers. We therefore generated a rabbit polyclonal Nogo receptor antibody from the leucine rich repeat number 9 domain of Nogo receptor polypeptide chain. Together with a commercially available polyclonal antibody specific for Nogo receptor, and in conjunction with double labelling immunofluorescence methods on crysections and cell cultures, Nogo receptor immunoreactivity was also observed in brain, spinal cord, and dorsal ganglia. In cellular populations, it was confined to neuronal cell bodies and their processes. Nogo receptor was localised on the surface of extending dorsal root ganglion intact axons and growth cones in live staining experiments. Nogo-A, an important axon growth inhibitory molecule and member of the reticulon family protein, was widely distributed in the mammalian brain, spinal cord, and dorsal root ganglia. Intense Nogo-A immunoreactivity was dete cted in oligodendrocyte cell bodies and their myelin sheaths in nerve fibre tracts of the central nervous system. Furthermore, numerous populations of neurons in the brain and spinal cord expressed Nogo-A to a variable extent in their cell bodies and neurites, suggesting additional, as-yet-unknown, functions of this protein. In cell culture, cytoplasmic staining with anti-Nogo-A antibody was observed after fixation oligodendrocytes and neurones, but intracellular structures that presumably represent endoplasmic reticulum were also strongly labelled in fibroblasts. These results confirm results obtained by other researchers with different set of antibodies. However, they also raise the question of the mechanism and circumstances under which the Nogo receptor interacts with Nogo-A, as this protein appears to be confined to the cytoplasm and can therefore not be expected to bind Nogo receptor on the axon surface. To investigate whether Nogo receptor is localised in neuronal lipid rafts, commercial and local antibodies specific for Nogo receptor, in conjunction with flotillin (a known lipid raft-associated protein) were used in double-immunofluorescence, co-immunoprecipitation and western blotting experiments. Results revealed substantial immunofluorescent colocalisation of Nogo receptor and flotillin in membranes of axons and PC-12 cells. Further more, extraction Nogo receptor antigen from rat brain using receptor bound protein-A sepharose beads, followed by probing with anti-flotillin antibody, established the link between lipid rafts and Nogo receptor.
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    Simvastatin enhances protection against Listeria monocytogenes infection in mice by counteracting Listeria-induced phagosomal escape
    (Public Library of Science, 2013) Parihar, Suraj P; Guler, Reto; Lang, Dirk M; Suzuki, Harukazu; Marais, A David; Brombacher, Frank
    Statins are well-known cholesterol lowering drugs targeting HMG-CoA-reductase, reducing the risk of coronary disorders and hypercholesterolemia. Statins are also involved in immunomodulation, which might influence the outcome of bacterial infection. Hence, a possible effect of statin treatment on Listeriosis was explored in mice. Statin treatment prior to subsequent L. monocytogenes infection strikingly reduced bacterial burden in liver and spleen (up to 100-fold) and reduced histopathological lesions. Statin-treatment in infected macrophages resulted in increased IL-12p40 and TNF-α and up to 4-fold reduced bacterial burden within 6 hours post infection, demonstrating a direct effect of statins on limiting bacterial growth in macrophages. Bacterial uptake was normal investigated in microbeads and GFP-expressing Listeria experiments by confocal microscopy. However, intracellular membrane-bound cholesterol level was decreased, as analyzed by cholesterol-dependent filipin staining and cellular lipid extraction. Mevalonate supplementation restored statin-inhibited cholesterol biosynthesis and reverted bacterial growth in Listeria monocytogenes but not in listeriolysin O (LLO)-deficient Listeria . Together, these results suggest that statin pretreatment increases protection against L. monocytogenes infection by reducing membrane cholesterol in macrophages and thereby preventing effectivity of the cholesterol-dependent LLO-mediated phagosomal escape of bacteria.