Oxidized phosphatidylcholines induce multiple functional defects in airway epithelial cells.

Oxidative stress is a hallmark of numerous airway diseases, contributing to extensive cell and tissue damage. Cell membranes and the airway mucosal lining are rich in phospholipids that are particularly susceptible to oxidative attack, producing bioactive molecules including oxidized phosphatidylcholines (OxPCs). With the recent discovery of elevated OxPCs in patients with asthma after allergen challenge, we hypothesized that OxPCs directly contribute to disease by inducing airway epithelial cell dysfunction. We found that OxPCs induced concentration-dependent cell stress and loss of viability in BEAS-2B and Calu-3 cell lines and primary human epithelial cells. These responses corresponded with significant epithelial barrier dysfunction, which was further compounded when combining OxPCs with an epithelial wound. OxPCs inhibited DNA synthesis and migration required to reestablish barrier function, but cells recovered if OxPCs were washed off soon after treatment. OxPCs induced generation of reactive oxygen species, lipid peroxidation, and mitochondrial dysfunction, raising the possibility that OxPCs cause pathological lipid metabolism in a self-propagating cycle. The oxidative stress induced by OxPCs could not be abrogated by putative OxPC receptor blockers, but partial recovery of barrier function, proliferation, and lipid peroxidation could be achieved with the antioxidant N-acetyl cysteine. In summary, we have identified OxPCs as a group of bioactive molecules that significantly impair multiple facets of epithelial cell function, consistent with pathological features of asthma. Further characterization of the mechanisms by which OxPCs affect epithelial cells could yield new insights into how oxidative stress contributes to the pathogenesis of airway disease.

Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis.

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

The influence of metallothionein treatment and treadmill running exercise on disease onset and survival in SOD1G93A amyotrophic lateral sclerosis mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1G93A familial ALS mice. Six-week-old female SOD1G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2-treated mice survived around 3% longer than vehicle-treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2-treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline-treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.

Aurora kinase B regulates axonal outgrowth and regeneration in the spinal motor neurons of developing zebrafish.

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration.

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.

The degree of astrocyte activation in multiple system atrophy is inversely proportional to the distance to α-synuclein inclusions.

Multiple system atrophy (MSA) exhibits widespread astrogliosis together with α-synuclein (α-syn) glial cytoplasmic inclusions (GCIs) in mature oligodendrocytes. We quantified astrocyte activation by morphometric analysis of MSA cases, and investigated the correlation to GCI proximity. Using Imaris software, we obtained "skinned" three-dimensional models of GFAP-positive astrocytes in MSA and control tissue (n=75) from confocal z-stacks and measured the astrocyte process length and thickness and radial distance to the GCI. Astrocytes proximal to GCI-containing oligodendrocytes (r<25µm) had significantly (p, 0.05) longer and thicker processes characteristic of activation than distal astrocytes (r>25µm), with a reciprocal linear correlation (m, 90µm(2)) between mean process length and radial distance to the nearest GCI (R(2), 0.7). In primary cell culture studies, α-syn addition caused ERK-dependent activation of rat astrocytes and perinuclear α-syn inclusions in mature (MOSP-positive) rat oligodendrocytes. Activated astrocytes were also observed in close proximity to α-syn deposits in a unilateral rotenone-lesion mouse model. Moreover, unilateral injection of MSA tissue-derived α-syn into the mouse medial forebrain bundle resulted in widespread neuroinflammation in the α-syn-injected, but not sham-injected hemisphere. Taken together, our data suggests that the action of localized concentrations of α-syn may underlie both astrocyte and oligodendrocyte MSA pathological features.

Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the motor system. Although the etiology of the disease is not fully understood, microglial activation and neuroinflammation are thought to play a role in disease progression. METHODS: We examined the immunohistochemical expression of two markers of microglial phenotype, the arginine-metabolizing enzymes inducible nitric oxide synthase (iNOS) and arginase1 (Arg1), in the spinal cord of a mouse model carrying an ALS-linked mutant human superoxide dismutase transgene (SOD1(G93A)) and in non-transgenic wild-type (WT) mice. Immunolabeling for iNOS and Arg1 was evaluated throughout disease progression (6 to 25 weeks), and correlated with body weight, stride pattern, wire hang duration and ubiquitin pathology. For microglia and motor neuron counts at each time point, SOD1(G93A) and WT animals were compared using an independent samples t-test. A Welch t-test correction was applied if Levene's test showed that the variance in WT and SOD1G93A measurements was substantially different. RESULTS: Disease onset, measured as the earliest change in functional parameters compared to non-transgenic WT mice, occurred at 14 weeks of age in SOD1(G93A) mice. The ventral horn of the SOD1(G93A) spinal cord contained more microglia than WT from 14 weeks onwards. In SOD1(G93A) mice, Arg1-positive and iNOS-positive microglia increased 18-fold and 7-fold, respectively, between 10 and 25 weeks of age (endpoint) in the lumbar spinal cord, while no increase was observed in WT mice. An increasing trend of Arg1- and iNOS-expressing microglia was observed in the cervical spinal cords of SOD1(G93A) mice. Additionally, Arg1-negative motor neurons appeared to selectively decline in the spinal cord of SOD1(G93A) mice, suggesting that Arg1 may have a neuroprotective function. CONCLUSIONS: This study suggests that the increase in spinal cord microglia occurs around and after disease onset and is preceded by cellular pathology. The results show that Arg1 and iNOS, thought to have opposing inflammatory properties, are upregulated in microglia during disease progression and that Arg1 in motor neurons may confer protection from disease processes. Further understanding of the neuroinflammatory response, and the Arg1/iNOS balance in motor neurons, may provide suitable therapeutic targets for ALS.

Labeling of cancer cells with magnetic nanoparticles for magnetic resonance imaging.

PURPOSE: The process of invasion and metastasis formation of tumor cells can be studied by following the migration of labeled cells over prolonged time periods. This report investigates the applicability of iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent for cell labeling. METHODS: γFe2 O3 nanoparticles prepared with direct flame spray pyrolysis are biofunctionalized with poly-l-lysine (PLL). The nanoparticles within the cells were observed with transmission electron microscopy, bright-field microscopy, and magnetorelaxometry. MRI of labeled cells suspended in agarose was used to estimate the detection limit. RESULTS: PLL-coated particles are readily taken up, stored in intracellular clusters, and gradually degraded by the cells. During cell division, the nanoparticle clusters are divided and split between daughter cells. The MRI detection limit was found to be 25 cells/mm(3) for R2*, and 70 cells/mm(3) for R2. The iron specificity, however, was higher for R2 images. Due to the degradation of intracellular γFe2 O3 to paramagnetic iron ions within 13 days, the R1, R2, and R2* contrast gradually decreased over this time period to approximately 50% of its initial value. CONCLUSIONS: These results suggest that PLL-coated γFe2 O3 nanoparticles can be used as an MRI contrast agent for long-term studies of cell migration. Magn Reson Med 71:1896-1905, 2014. © 2013 Wiley Periodicals, Inc.

Airway smooth muscle in asthma: linking contraction and mechanotransduction to disease pathogenesis and remodelling.

Asthma is an obstructive airway disease, with a heterogeneous and multifactorial pathogenesis. Although generally considered to be a disease principally driven by chronic inflammation, it is becoming increasingly recognised that the immune component of the pathology poorly correlates with the clinical symptoms of asthma, thus highlighting a potentially central role for non-immune cells. In this context airway smooth muscle (ASM) may be a key player, as it comprises a significant proportion of the airway wall and is the ultimate effector of acute airway narrowing. Historically, the contribution of ASM to asthma pathogenesis has been contentious, yet emerging evidence suggests that ASM contractile activation imparts chronic effects that extend well beyond the temporary effects of bronchoconstriction. In this review article we describe the effects that ASM contraction, in combination with cellular mechanotransduction and novel contraction-inflammation synergies, contribute to asthma pathogenesis. Specific emphasis will be placed on the effects that ASM contraction exerts on the mechanical properties of the airway wall, as well as novel mechanisms by which ASM contraction may contribute to more established features of asthma such as airway wall remodelling.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations.

We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn(2+) accumulation. Comparison between two dialysis conditions (standard; 20 min, brief; 2 min) by standard whole-cell clamp revealed a high vulnerability of intracellular Zn(2+) buffers to intracellular dialysis. Thus, low concentrations of zinc-pyrithione generated robust responses in neurons with standard dialysis, but signals were smaller in neurons with short dialysis. Release from oxidation-sensitive Zn(2+) pools was reduced by standard dialysis, when compared with responses in neurons with brief dialysis. The dialysis effects were partly reversed by inclusion of recombinant metallothionein-3 in the dialysis solution. These findings suggested that extensive dialysis could be exploited for selective detection of transmembrane Zn(2+) influx. Different dialysis conditions were then used to probe responses to synaptic stimulation. Under standard dialysis conditions, synaptic stimuli generated significant FluoZin-3 signals in wild-type (WT) preparations, but responses were almost absent in preparations lacking vesicular Zn(2+) (ZnT3-KO). In contrast, under brief dialysis conditions, intracellular Zn(2+) transients were very similar in WT and ZnT3-KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn(2+) accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn(2+) accumulation mechanisms.

Airway contractility and remodeling: links to asthma symptoms.

Respiratory symptoms are largely caused by obstruction of the airways. In asthma, airway narrowing mediated by airway smooth muscle (ASM) contraction contributes significantly to obstruction. The spasmogens produced following exposure to environmental triggers, such as viruses or allergens, are initially responsible for ASM activation. However, the extent of narrowing of the airway lumen due to ASM shortening can be influenced by many factors and it remains a real challenge to decipher the exact role of ASM in causing asthmatic symptoms. Innovative tools, such as the forced oscillation technique, continue to develop and have been proven useful to assess some features of ASM function in vivo. Despite these technologic advances, it is still not clear whether excessive narrowing in asthma is driven by ASM abnormalities, by other alterations in non-muscle factors or simply because of the overexpression of spasmogens. This is because a multitude of forces are acting on the airway wall, and because not only are these forces constantly changing but they are also intricately interconnected. To counteract these limitations, investigators have utilized in vitro and ex vivo systems to assess and compare asthmatic and non-asthmatic ASM contractility. This review describes: 1- some muscle and non-muscle factors that are altered in asthma that may lead to airway narrowing and asthma symptoms; 2- some technologies such as the forced oscillation technique that have the potential to unveil the role of ASM in airway narrowing in vivo; and 3- some data from ex vivo and in vitro methods that probe the possibility that airway hyperresponsiveness is due to the altered environment surrounding the ASM or, alternatively, to a hypercontractile ASM phenotype that can be either innate or acquired.

Models to study airway smooth muscle contraction in vivo, ex vivo and in vitro: implications in understanding asthma.

Asthma is a chronic obstructive airway disease characterised by airway hyperresponsiveness (AHR) and airway wall remodelling. The effector of airway narrowing is the contraction of airway smooth muscle (ASM), yet the question of whether an inherent or acquired dysfunction in ASM contractile function plays a significant role in the disease pathophysiology remains contentious. The difficulty in determining the role of ASM lies in limitations with the models used to assess contraction. In vivo models provide a fully integrated physiological response but ASM contraction cannot be directly measured. Ex vivo and in vitro models can provide more direct assessment of ASM contraction but the loss of factors that may modulate ASM responsiveness and AHR, including interaction between multiple cell types and disruption of the mechanical environment, precludes a complete understanding of the disease process. In this review we detail key advantages of common in vivo, ex vivo and in vitro models of ASM contraction, as well as emerging tissue engineered models of ASM and whole airways. We also highlight important findings from each model with respect to the pathophysiology of asthma.

Metallothionein promotes regenerative axonal sprouting of dorsal root ganglion neurons after physical axotomy.

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism.

Transcriptional insights on the regenerative mechanics of axotomized neurons in vitro.

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ± 1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.

Airways dilate to simulated inspiratory but not expiratory manoeuvres.

In a healthy human, deep inspirations produce bronchodilation of contracted airways, which probably occurs due to the transient distension of the airway smooth muscle (ASM). We hypothesised that deep expiratory manoeuvres also produce bronchodilation due to transient airway wall and ASM compression. We used porcine bronchial segments to assess the effects of deep inspirations, and maximal and partial expiration (submaximal) on airway calibre. Respiratory manoeuvres were simulated by varying transmural pressure using a hydrostatic pressure column: deep inspiration 5 to 30 cmH(2)O, maximal expiration 30 to -15 cmH(2)O, partial expiration 10 to -15 cmH(2)O; amidst a background of tidal oscillations, 5 to 10 cmH(2)O at 0.25 Hz. Changes in luminal cross-sectional area in carbachol-contracted airways were measured using video endoscopy. Deep inspirations produce an immediate bronchodilation (∼40-60%, p=0.0076) that lasts for up to 1 min (p=0.0479). In comparison, after maximal expiration there was no immediate change in airway calibre; however, a delayed bronchodilatory response was observed from 4 s after the manoeuvre (p=0.0059) and persisted for up to 3 min (p=0.0182). Partial expiration had little or no effect or airway calibre. The results observed demonstrate that the airway wall dilates to deep inspiration manoeuvres but is unresponsive to deep expiratory manoeuvres.

Cytokines and olfactory bulb microglia in response to bacterial challenge in the compromised primary olfactory pathway.

BACKGROUND: The primary olfactory pathway is a potential route through which microorganisms from the periphery could potentially access the central nervous system. Our previous studies demonstrated that if the olfactory epithelium was damaged, bacteria administered into the nasal cavity induced nitric oxide production in olfactory ensheathing cells. This study investigates the cytokine profile of olfactory tissues as a consequence of bacterial challenge and establishes whether or not the bacteria are able to reach the olfactory bulb in the central nervous system. METHODS: The olfactory epithelium of C57BL/6 mice was damaged by unilateral Triton X-100 nasal washing, and Staphylococcus aureus was administered ipsilaterally 4 days later. Olfactory mucosa and bulb were harvested 6 h, 24 h and 5 days after inoculation and their cytokine profile compared to control tissues. The fate of S. aureus and the response of bulbar microglia were examined using fluorescence microscopy and transmission electron microscopy. RESULTS: In the olfactory mucosa, administered S. aureus was present in supporting cells of the olfactory epithelium, and macrophages and olfactory nerve bundles in the lamina propria. Fluorescein isothiocyanate-conjugated S. aureus was observed within the olfactory mucosa and bulb 6 h after inoculation, but remained restricted to the peripheral layers up to 5 days later. At the 24-h time point, the level of interleukin-6 (IL-6) and tumour necrosis factor-α in the compromised olfactory tissues challenged with bacteria (12,466 ± 956 pg/ml and 552 ± 193 pg/ml, respectively) was significantly higher than that in compromised olfactory tissues alone (6,092 ± 1,403 pg/ml and 80 ± 2 pg/ml, respectively). Immunohistochemistry confirmed that IL-6 was present in several cell types including olfactory ensheathing cells and mitral cells of the olfactory bulb. Concurrently, there was a 4.4-, 4.5- and 2.8-fold increase in the density of iNOS-expressing cells in the olfactory mucosa, olfactory nerve and glomerular layers combined, and granule layer of the olfactory bulb, respectively. CONCLUSIONS: Bacteria are able to penetrate the immunological defence of the compromised olfactory mucosa and infiltrate the olfactory bulb within 6 h even though a proinflammatory profile is mounted. Activated microglia may have a role in restricting bacteria to the outer layers of the olfactory bulb.

Burn injury has a systemic effect on reinnervation of skin and restoration of nociceptive function.

Burn injury can lead to abnormal sensory function at both the injury and at distant uninjured sites. Here, we used a mouse model to investigate return of nociceptive function and reinnervation of the skin at the wound and uninjured distant sites following a 3% total burn surface area full-thickness burn injury. We have previously shown that topical application of zinc-metallothionein-IIA (Zn(7) -MT-IIA) accelerates healing following burn injury, and here, we investigated the potential of Zn(7) -MT-IIA to enhance reinnervation and sensory recovery. In all burn-injured animals, there was a significant reduction in nociceptive responses (Semmes-Weinstein filaments) at locations near and distant to the wound up to 8 weeks following injury. Cutaneous nerve reinnervation (assessed using protein gene product 9.5 immunohistochemistry) of the wound center was slow in the epidermis but rapid in the dermis. In the dermis, nerves subsequently degenerated both at the wound center and in distant uninjured areas. In contrast, epidermal nerve densities in the distant uninjured areas returned to normal, uninjured levels. Zn(7) -MT-IIA did not influence return of nociceptive function nor reinnervation. We conclude that burn injury compromises nociceptive function and nerve regeneration both at the injury site and systemically; thus, therapies in addition to Zn(7) -MT-IIA should be explored to return normal sensory function.

Olfactory ensheathing cells moderate nuclear factor kappaB translocation in astrocytes.

Nuclear factor kappaB (NFκB) is a key transcriptional regulator of inflammatory genes. We investigated the modulatory effects of olfactory ensheathing cells (OECs), microglia and meningeal fibroblasts on translocation of NFκB to astrocyte nuclei. The percentage of activated astrocytes in co-cultures with OECs was significantly less than for co-cultures with microglia (p<0.001) and fibroblasts (p<0.05). Phorbol myristate acetate (PMA) and calcium ionophore stimulation of p65 NFκB translocation to nuclei provided an in vitro model of astrocyte inflammatory activation. Soluble factors released by OECs significantly moderated the astrocytic NFκB translocation induced by either PMA/calcium ionophore or microglia-derived factors (p<0.001). Insulin-like growth factor-1 may contribute to these effects, since it is expressed by OECs and also significantly moderated the astrocytic NFκB translocation (p<0.05), albeit insufficiently to fully account for the OEC-induced moderation (p<0.01). Olfactory ensheathing cells significantly moderated the increased transcription of the pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor in the activated astrocytes (p<0.01). These results suggest that transplanted OECs could improve neural repair after CNS injury by moderating astrocyte activation.

The maternal-fetal transfer of passive immunity as a mechanism of transplacental nanoparticle drug delivery for prenatal therapies.

Nanoparticles administered into the maternal circulation and across the placenta are a potential clinical therapy to treat congenital diseases. The mechanism by which nanoparticles can safely cross the placenta for targeted drug delivery to the fetus remains poorly understood. We demonstrate that the maternal-fetal transfer of passive immunity through the neonatal Fc Receptor (FcRn) can induce the transplacental transfer of chitosan nanoparticles modifed with IgG antibodies (414 ± 27 nm). The transfer of FITC-tagged IgG-modified chitosan nanoparticles was 2.8 times higher (p = 0.0264) compared to similarly-sized unmodified chitosan nanoparticles (375 ± 17 nm). Co-administration of free IgG competitively diminished the transplacental transfer of IgG-modified nanoparticles, yet unmodified nanoparticles remained unaffected. Colocalization of the FcRn and the IgG-modified chitosan nanoparticles were observed with confocal microscopy. Barrier function before and after nanoparticle administration remained intact as determined by TEER (75-79 Ω cm2) and immmunofluorescence of ZO-1 tight junction proteins. The results provide insight into the clinical applications of nanoparticles for prenatal therapies using the mechanism of the maternal-fetal transfer of passive immunity.