Fractalkine isoforms differentially regulate microglia-mediated inflammation and enhance visual function in the diabetic retina.

Diabetic retinopathy (DR) affects about 200 million people worldwide, causing leakage of blood components into retinal tissues, leading to activation of microglia, the resident phagocytes of the retina, promoting neuronal and vascular damage. The microglial receptor, CX3CR1, binds to fractalkine (FKN), an anti-inflammatory chemokine that is expressed on neuronal membranes (mFKN), and undergoes constitutive cleavage to release a soluble domain (sFKN). Deficiencies in CX3CR1 or FKN showed increased microglial activation, inflammation, vascular damage, and neuronal loss in experimental mouse models. To understand the mechanism that regulates microglia function, recombinant adeno-associated viral vectors (rAAV) expressing mFKN or sFKN were delivered to intact retinas prior to diabetes. High-resolution confocal imaging and mRNA-seq were used to analyze microglia morphology and markers of expression, neuronal and vascular health, and inflammatory mediators. We confirmed that prophylactic intra-vitreal administration of rAAV expressing sFKN (rAAV-sFKN), but not mFKN (rAAV-mFKN), in FKNKO retinas provided vasculo- and neuro-protection, reduced microgliosis, mitigated inflammation, improved overall optic nerve health by regulating microglia-mediated inflammation, and prevented fibrin(ogen) leakage at 4 weeks and 10 weeks of diabetes induction. Moreover, administration of sFKN improved visual acuity. Our results elucidated a novel intervention via sFKN gene therapy that provides an alternative pathway to implement translational and therapeutic approaches, preventing diabetes-associated blindness.

Therapeutic Delivery of Soluble Fractalkine Ameliorates Vascular Dysfunction in the Diabetic Retina.

Diabetic retinopathy (DR)-associated vision loss is a devastating disease affecting the working-age population. Retinal pathology is due to leakage of serum components into retinal tissues, activation of resident phagocytes (microglia), and vascular and neuronal damage. While short-term interventions are available, they do not revert visual function or halt disease progression. The impact of microglial inflammatory responses on the neurovascular unit remains unknown. In this study, we characterized microglia-vascular interactions in an experimental model of DR. Early diabetes presents activated retinal microglia, vascular permeability, and vascular abnormalities coupled with vascular tortuosity and diminished astrocyte and endothelial cell-associated tight-junction (TJ) and gap-junction (GJ) proteins. Microglia exclusively bind to the neuronal-derived chemokine fractalkine (FKN) via the CX3CR1 receptor to ameliorate microglial activation. Using neuron-specific recombinant adeno-associated viruses (rAAVs), we therapeutically overexpressed soluble (sFKN) or membrane-bound (mFKN) FKN using intra-vitreal delivery at the onset of diabetes. This study highlights the neuroprotective role of rAAV-sFKN, reducing microglial activation, vascular tortuosity, fibrin(ogen) deposition, and astrogliosis and supporting the maintenance of the GJ connexin-43 (Cx43) and TJ zonula occludens-1 (ZO-1) molecules. The results also show that microglia-vascular interactions influence the vascular width upon administration of rAAV-sFKN and rAAV-mFKN. Administration of rAAV-sFKN improved visual function without affecting peripheral immune responses. These findings suggest that overexpression of rAAV-sFKN can mitigate vascular abnormalities by promoting glia-neural signaling. sFKN gene therapy is a promising translational approach to reverse vision loss driven by vascular dysfunction.

Concentration and proteolysis of CX3CL1 may regulate the microglial response to CX3CL1.

Fractalkine (FKN) is a membrane-bound chemokine that can be cleaved by proteases such as ADAM 10, ADAM 17, and cathepsin S to generate soluble fragments. Studies using different forms of the soluble FKN yield conflicting results in vivo. These observations prompted us to investigate the function and pharmacology of two commonly used isoforms of FKN, a human full-length soluble FKN (sFKN), and a human chemokine domain only FKN (cdFKN). Both are prevalent in the literature and are often assumed to be functionally equivalent. We observed that recombinant sFKN and cdFKN exhibit similar potencies in a cell-based cAMP assay, but binding affinity for CX3CR1 was modestly different. There was a 10-fold difference in potency between sFKN and cdFKN when assessing their ability to stimulate ß-arrestin recruitment. Interestingly, high concentrations of FKN, regardless of cleavage variant, were ineffective at reducing pro-inflammatory microglial activation and may induce a pro-inflammatory response. This effect was observed in mouse and rat primary microglial cells as well as microglial cell lines. The inflammatory response was exacerbated in aged microglia, which is known to exhibit age-related inflammatory phenotypes. We observed the same effects in Cx3cr1-/- primary microglia and therefore speculate that an alternative FKN receptor may exist. Collectively, these data provide greater insights into the function and pharmacology of these common FKN reagents, which may clarify conflicting reports and urge greater caution in the selection of FKN peptides for use in in vitro and in vivo studies and the interpretation of results obtained using these differing peptides.

A novel Reelin construct, R36, recovered behavioural deficits in the heterozygous reeler mouse.

Reelin, a large extracellular glycoprotein, plays a critical role in prenatal brain development and postnatally in synaptic plasticity, learning and memory. Dysregulation of Reelin signalling has been implicated in several neuropsychiatric disorders including schizophrenia, autism, depression and Alzheimer's disease. Previous studies have demonstrated that Reelin's central fragment, R3456, binds to ApoER2, inducing ApoER2 clustering and subsequent intracellular signalling. We previously reported the development of a novel luciferase complementation assay, which we used to demonstrate that R3456 can lead to ApoER2 receptor dimerization. Using this same assay, we explored various smaller fragments and combinations from R3456, and we identified a construct of repeats 3 and 6 (R36), which could still elicit equivalent receptor dimerization. The purpose of this study was to test R36 for biological effects in vitro and in vivo. We show that R36 was capable of initiating intracellular signalling in primary neuronal cultures. In addition, we demonstrate that a single intracerebroventricular injection of R36 protein into a model of Reelin deficiency, the heterozygous reeler mice, can significantly improve cognition. These data support a role for the new construct R36 to enhance the Reelin pathway, and the future possibility of exploring gene therapy approaches with R36 in diseases characterized by reduced levels of Reelin.

Lung infection by Pseudomonas aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

BACKGROUND: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. METHODS: Lung infection in mice was induced by instilling Pseudomonas aeruginosa (PA) intratracheally. We determined bacterial colonization in tissue, microvascular leakage, expression of cytokines and leukocyte infiltration into the brain. RESULTS: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 h and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + CD45+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) and adherens junction (AJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. CONCLUSIONS: Lung bacterial infection is associated with BBB disruption and behavioral changes, which are mediated by systemic cytokine release.

Recovery of Angelman syndrome rat deficits with UBE3A protein supplementation.

We recently generated a novel Angelman syndrome (AS) rat model with a complete Ube3a gene deletion, that recapitulates the loss of UBE3A protein and shows cognitive and EEG deficits. We also recently published the identification of extracellular UBE3A protein within the brain using microdialysis. Here we explored the effects of supplementation of exogenous UBE3A protein to hippocampal slices and intrahippocampal injection of AS rats. We report that the AS rat model demonstrates deficits in hippocampal long-term potentiation (LTP) which can be recovered with the application of exogenous UBE3A protein. Furthermore, injection of recombinant UBE3A protein into the hippocampus of the AS rat can rescue the associative learning and memory deficits seen in the fear conditioning task. These data suggest that extracellular UBE3A protein may play a role in synaptic function, LTP induction and hippocampal-dependent memory formation.

Overexpression of human wtTDP-43 causes impairment in hippocampal plasticity and behavioral deficits in CAMKII-tTa transgenic mouse model.

AIMS: The current study utilizes the adeno-associated viral gene transfer system in the CAMKIIα-tTA mouse model to overexpress human wild type TDP-43 (wtTDP-43) and α-synuclein (α-Syn) proteins. The co-existence of these proteins is evident in the pathology of neurodegenerative disorders such as frontotemporal lobar degeneration (FTLD), Parkinson disease (PD), and dementia with Lewy bodies (DLB). METHODS: The novel bicistronic recombinant adeno-associated virus (rAAV) serotype 9 drives wtTDP-43 and α-Syn expression in the hippocampus via "TetO" CMV promoter. Behavior, electrophysiology, and biochemical and histological assays were used to validate neuropathology. RESULTS: We report that overexpression of wtTDP-43 but not α-Syn contributes to hippocampal CA2-specific pyramidal neuronal loss and overall hippocampal atrophy. Further, we report a reduction of hippocampal long-term potentiation and decline in learning and memory performance of wtTDP-43 expressing mice. Elevated wtTDP-43 levels induced selective degeneration of Purkinje cell protein 4 (PCP-4) positive neurons while both wtTDP-43 and α-Syn expression reduced subsets of the glutamate receptor expression in the hippocampus. CONCLUSIONS: Overall, our findings suggest the significant vulnerability of hippocampal neurons toward elevated wtTDP-43 levels possibly via PCP-4 and GluR-dependent calcium signaling pathways. Further, we report that wtTDP-43 expression induced selective CA2 subfield degeneration, contributing to the deterioration of the hippocampal-dependent cognitive phenotype.

T cell infiltration and upregulation of MHCII in microglia leads to accelerated neuronal loss in an α-synuclein rat model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most prevalent movement disorder characterized by up to 80% loss of dopamine (DA) neurons and accumulation of Lewy body deposits composed of α-synuclein (α-syn). Accumulation of α-syn is associated with microglial activation, leading to a pro-inflammatory environment linked with the pathogenesis of PD. Along with microglia, CD4 and CD8 T cells are observed in SNpc. The contribution of T-cells to PD development remains unclear with studies demonstrating that they may mediate neurodegeneration or act in a neuroprotective manner. METHODS: Here, we assessed the contribution of T cells to PD neurodegeneration using an adeno-associated virus (AAV) coding human wild-type α-syn or GFP injected into the substantia nigra pars compacta (SNpc) in T cell deficient (athymic nude) and T cell competent (heterozygous) rats. The rats were behaviorally assessed with cylinder test to test paw bias. Following behavior testing, brains were collected and analyzed for markers of dopamine neuron, microglial activation, T cells, and α-syn expression. RESULTS: Injection of AAV9-α-syn unilaterally into the SN of T cell competent rats resulted in a significant paw bias in comparison to the controls at 60 days post-injection. Conversely, T cell-deficient rats injected with AAV9-α-syn showed no deficit in paw bias. As expected, injected T cell competent rats demonstrated a significant increase in microglial activation (MHCII staining) as well as significant dopaminergic neuron loss. In contrast, the T cell-deficient counterparts did not show a significant increase in microglial activation or significant neuron loss compared to the control animals. We also observed CD4 and CD8 T cells in SNpc following microglial MHCII expression and dopaminergic neuron loss. The time course of T cell entry correlates with upregulation of MHCII and the peak loss of TH+ cells in the SNpc. CONCLUSION: These data demonstrate that T cell infiltration and microglial upregulation of MHCII are involved in α-synuclein-mediated DA neuron loss in this rat model of PD.

Two forms of CX3CL1 display differential activity and rescue cognitive deficits in CX3CL1 knockout mice.

BACKGROUND: Fractalkine (CX3CL1; FKN) is a chemokine expressed by neurons that mediates communication between neurons and microglia. By regulating microglial activity, CX3CL1 can mitigate the damaging effects of chronic microglial inflammation within the brain, a state that plays a major role in aging and neurodegeneration. CX3CL1 is present in two forms, a full-length membrane-bound form and a soluble cleaved form (sFKN), generated by a disintegrin and metalloproteinase (ADAM) 10 or 17. Levels of sFKN decrease with aging, which could lead to enhanced inflammation, deficits in synaptic remodeling, and subsequent declines in cognition. Recently, the idea that these two forms of CX3CL1 may display differential activities within the CNS has garnered increased attention, but remains unresolved. METHODS: Here, we assessed the consequences of CX3CL1 knockout (CX3CL1-/-) on cognitive behavior as well as the functional rescue with the two different forms of CX3CL1 in mice. CX3CL1-/- mice were treated with adeno-associated virus (AAV) expressing either green fluorescent protein (GFP), sFKN, or an obligate membrane-bound form of CX3CL1 (mFKN) and then subjected to behavioral testing to assess cognition and motor function. Following behavioral analysis, brains were collected and analyzed for markers of neurogenesis, or prepared for electrophysiology to measure long-term potentiation (LTP) in hippocampal slices. RESULTS: CX3CL1-/- mice showed significant deficits in cognitive tasks for long-term memory and spatial learning and memory in addition to demonstrating enhanced basal motor performance. These alterations correlated with deficits in both hippocampal neurogenesis and LTP. Treatment of CX3CL1-/- mice with AAV-sFKN partially corrected changes in both cognitive and motor function and restored neurogenesis and LTP to levels similar to wild-type animals. Treatment with AAV-mFKN partially restored spatial learning and memory in CX3CL1-/- mice, but did not rescue long-term memory, or neurogenesis. CONCLUSIONS: These results are the first to demonstrate that CX3CL1 knockout causes significant cognitive deficits that can be rescued by treatment with sFKN and only partially rescued with mFKN. This suggests that treatments that restore signaling of soluble forms of CX3CL1 may be a viable therapeutic option for aging and disease.

TDP-43 mediated blood-brain barrier permeability and leukocyte infiltration promote neurodegeneration in a low-grade systemic inflammation mouse model.

BACKGROUND: Neuronal cytoplasmic inclusions containing TAR DNA-binding protein 43 (TDP-43) are a neuropathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's Disease (AD). Emerging evidence also indicates that systemic inflammation may be a contributor to the pathology progression of these neurodegenerative diseases. METHODS: To investigate the role of systemic inflammation in the progression of neuronal TDP-43 pathology, AAV9 particles driven by the UCHL1 promoter were delivered to the frontal cortex of wild-type aged mice via intracranial injections to overexpress TDP-43 or green fluorescent protein (GFP) in corticospinal motor neurons. Animals were then subjected to a low-dose (500 µg/kg) intraperitoneal E. coli lipopolysaccharide (LPS) administration challenge for 2 weeks to mimic a chronically altered low-grade systemic inflammatory state. Mice were then subjected to neurobehavioral studies, followed by biochemical and immunohistochemical analyses of the brain tissue. RESULTS: In the present study, we report that elevated neuronal TDP-43 levels induced microglial and astrocytic activation in the cortex of injected mice followed by increased RANTES signaling. Moreover, overexpression of TDP-43 exerted abundant mouse immunoglobulin G (IgG), CD3, and CD4+ T cell infiltration as well as endothelial and pericyte activation suggesting increased blood-brain barrier permeability. The BBB permeability in TDP-43 overexpressing brains yielded the frontal cortex vulnerable to the systemic inflammatory response following LPS treatment, leading to marked neutrophil infiltration, neuronal loss, reduced synaptosome-associated protein 25 (SNAP-25) levels, and behavioral impairments in the radial arm water maze (RAWM) task. CONCLUSIONS: These results reveal a novel role for TDP-43 in BBB permeability and leukocyte recruitment, indicating complex intermolecular interactions between an altered systemic inflammatory state and pathologically prone TDP-43 protein to promote disease progression.

Neuroinflammation and fractalkine signaling in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder, and the most common form of dementia. As the understanding of AD has progressed, it is now believed that AD is an amyloid-initiated tauopathy with neuroinflammation serving as the link between amyloid deposition, tau pathology, and neurodegeneration. As microglia are the main immune effectors in the central nervous system, they have been the focus of attention in studies investigating the neuroinflammatory component of AD. Therefore, recent work has focused on immunomodulators, which can alter microglial activation without suppressing activity, as potential therapeutics for AD. Fractalkine (CX3CL1; FKN), a unique chemokine with a one-to-one relationship with its receptor, signals through its cognate receptor (CX3CR1) to reduce expression of pro-inflammatory genes in activated microglia. Disrupting FKN signaling has opposing effects on the two hallmark pathologies of AD, but over-expressing a soluble FKN has been shown to reduce tau pathology while not altering amyloid pathology. Recently, differential signaling has been reported when comparing two cleavage variants of soluble FKN. These differential effects may explain recent studies reporting seemingly conflicting results regarding the effect of FKN over expression on AD pathologies.

Sustained Arginase 1 Expression Modulates Pathological Tau Deposits in a Mouse Model of Tauopathy.

Tau accumulation remains one of the closest correlates of neuronal loss in Alzheimer's disease. In addition, tau associates with several other neurodegenerative diseases, collectively known as tauopathies, in which clinical phenotypes manifest as cognitive impairment, behavioral disturbances, and motor impairment. Polyamines act as bivalent regulators of cellular function and are involved in numerous biological processes. The regulation of the polyamines system can become dysfunctional during disease states. Arginase 1 (Arg1) and nitric oxide synthases compete for l-arginine to produce either polyamines or nitric oxide, respectively. Herein, we show that overexpression of Arg1 using adeno-associated virus (AAV) in the CNS of rTg4510 tau transgenic mice significantly reduced phospho-tau species and tangle pathology. Sustained Arg1 overexpression decreased several kinases capable of phosphorylating tau, decreased inflammation, and modulated changes in the mammalian target of rapamycin and related proteins, suggesting activation of autophagy. Arg1 overexpression also mitigated hippocampal atrophy in tau transgenic mice. Conversely, conditional deletion of Arg1 in myeloid cells resulted in increased tau accumulation relative to Arg1-sufficient mice after transduction with a recombinant AAV-tau construct. These data suggest that Arg1 and the polyamine pathway may offer novel therapeutic targets for tauopathies.

Fractalkine over expression suppresses α-synuclein-mediated neurodegeneration.

In Parkinson's disease, α-synuclein is known to activate microglia and this activation has been proposed as one of the mechanisms of neurodegeneration. There are several signals produced by neurons that have an anti-inflammatory action on microglia, including CX3CL1 (fractalkine). We have shown that a soluble form of CX3CL1 is required to reduce neuron loss in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and that fractalkine agonism can reduce neuron loss in a 6-hydroxydopamine lesion model. Here, we show that fractalkine can reduce α-synuclein-mediated neurodegeneration in rats. Rats that received fractalkine showed abrogated loss of tyrosine hydroxylase and Neu-N staining. This was replicated in animals where we expressed fractalkine from astrocytes with the glial fibrillary acid protein (GFAP) promoter. Interestingly, we did not observe a reduction in MHCII expression suggesting that soluble fractalkine is likely altering the microglial state to a more neuroprotective one rather than reducing antigen presentation.

Utility of sequencing the erm(41) gene in isolates of Mycobacterium abscessus subsp. abscessus with low and intermediate clarithromycin MICs.

The erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of ≥8 µg/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of ≤8 µg/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of ≥16 µg/ml, including ATCC 19977T, revealed 10 sequevars. The sequence in ATCC 19977T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 µg/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of ≤2 (80%), 4 (10%), and 8 (10%) µg/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from ≤2 to ≤4 µg/ml and that isolates with an MIC of 8 µg/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.

Absence of a functional erm gene in isolates of Mycobacterium immunogenum and the Mycobacterium mucogenicum group, based on in vitro clarithromycin susceptibility.

Macrolide resistance has been linked to the presence of a functional erythromycin ribosomal methylase (erm) gene in most species of pathogenic rapidly growing mycobacteria (RGM). For these Mycobacterium isolates, extended incubation in clarithromycin is necessary to determine macrolide susceptibility. In contrast, the absence of a detectable erm gene in isolates of M. chelonae, M. senegalense, and M. peregrinum and a nonfunctional erm gene in M. abscessus subsp. massiliense and 15% to 20% of M. abscessus subsp. abscessus isolates renders these species intrinsically macrolide susceptible. Not all RGM species have been screened for the presence of an erm gene, including the Mycobacterium mucogenicum group (M. mucogenicum, M. phocaicum, and M. aubagnense) and Mycobacterium immunogenum. A total of 356 isolates of these two pathogenic RGM taxa from two reference laboratories (A.R.U.P. Reference Laboratories and the Mycobacteria/Nocardia Laboratory at the University of Texas Health Science Center at Tyler) underwent clarithromycin susceptibility testing with readings at 3 to 5 days and 14 days. Only 13 of the 356 isolates had resistant clarithromycin MICs at initial extended MIC readings, and repeat values on all available isolates were ≤2 µg/ml. These studies suggest that these two additional RGM groups do not harbor functional erm genes and, like M. chelonae, do not require extended clarithromycin susceptibility testing. We propose to the Clinical Laboratory and Standards Institute that isolates belonging to these above-mentioned six rapidly growing mycobacterial groups based on molecular identification with no known functional erm genes undergo only 3 to 5 days of susceptibility testing (to exclude mutational resistance).

Nanomedicine in the ROS-mediated pathophysiology: Applications and clinical advances.

Reactive oxygen species (ROS) are important in regulating normal cell physiological functions, but when produced in excess lead to the augmented pathogenesis of various diseases. Among these, ischemia reperfusion injury, Alzheimer's disease and rheumatoid arthritis are particularly important. Since ROS can be counteracted by a variety of antioxidants, natural and synthetic antioxidants have been developed. However, due to the ubiquitous production of ROS in living systems, poor in vivo efficiency of these agents and lack of target specificity, the current clinical modalities to treat oxidative stress damage are limited. Advances in the developing field of nanomedicine have yielded nanoparticles that can prolong antioxidant activity, and target specificity of these agents. This article reviews recent advances in antioxidant nanoparticles and their applications to manage oxidative stress-mediated diseases. FROM THE CLINICAL EDITOR: Production of reactive oxygen species (ROS) is a purely physiological process in many disease conditions. However, excessive and uncontrolled production will lead to oxidative stress and further tissue damage. Advances in nanomedicine have provided many novel strategies to try to combat and counteract ROS. In this review article, the authors comprehensively highlighted the current status and future developments in using nanotechnology for providing novel therapeutic options in this field.

Reactivities of substituted α-phenyl-N-tert-butyl nitrones.

In this work, a series of α-phenyl-N-tert-butyl nitrones bearing one, two, or three substituents on the tert-butyl group was synthesized. Cyclic voltammetry (CV) was used to investigate their electrochemical properties and showed a more pronounced substituent effect for oxidation than for reduction. Rate constants of superoxide radical (O2(•-)) reactions with nitrones were determined using a UV-vis stopped-flow method, and phenyl radical (Ph(•)) trapping rate constants were measured by EPR spectroscopy. The effect of N-tert-butyl substitution on the charge density and electron density localization of the nitronyl carbon as well as on the free energies of nitrone reactivity with O2(•-) and HO2(•) were computationally rationalized at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory. Theoretical and experimental data showed that the rates of the reaction correlate with the nitronyl carbon charge density, suggesting a nucleophilic nature of O2(•-) and Ph(•) addition to the nitronyl carbon atom. Finally, the substituent effect was investigated in cell cultures exposed to hydrogen peroxide and a correlation between the cell viability and the oxidation potential of the nitrones was observed. Through a combination of computational methodologies and experimental methods, new insights into the reactivity of free radicals with nitrone derivatives have been proposed.

Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria.

Within the past 10 years, treatment and diagnostic guidelines for nontuberculous mycobacteria have been recommended by the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA). Moreover, the Clinical and Laboratory Standards Institute (CLSI) has published and recently (in 2011) updated recommendations including suggested antimicrobial and susceptibility breakpoints. The CLSI has also recommended the broth microdilution method as the gold standard for laboratories performing antimicrobial susceptibility testing of nontuberculous mycobacteria. This article reviews the laboratory, diagnostic, and treatment guidelines together with established and probable drug resistance mechanisms of the nontuberculous mycobacteria.

Adenylyl cyclase 2 expression and function in neurological diseases.

Adenylyl cyclases (Adcys) catalyze the formation of cAMP, a secondary messenger essential for cell survival and neurotransmission pathways in the CNS. Adcy2, one of ten Adcy isoforms, is highly expressed in the CNS. Abnormal Adcy2 expression and mutations have been reported in various neurological disorders in both rodents and humans. However, due to the lack of genetic tools, loss-of-function studies of Adcy2 are scarce. In this review, we summarize recent findings on Adcy2 expression and function in neurological diseases. Specifically, we first introduce the biochemistry, structure, and function of Adcy2 briefly. Next, the expression and association of Adcy2 in human patients and rodent models of neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), psychiatric disorders (Tourette syndrome, schizophrenia, and bipolar disorder), and other neurological conditions (stress-associated disorders, stroke, epilepsy, and Lesch-Nyhan Syndrome) are elaborated. Furthermore, we discuss the pros and cons of current studies as well as key questions that need to be answered in the future. We hope to provide a focused review on Adcy2 that promotes future research in the field.

The soluble isoform of CX3CL1 is necessary for neuroprotection in a mouse model of Parkinson's disease.

The chemokine CX3CL1/fractalkine is expressed by neurons as a transmembrane-anchored protein that can be cleaved to yield a soluble isoform. However, the roles for these two types of endogenous CX3CL1 in neurodegenerative pathophysiology remain elusive. As such, it has been difficult to delineate the function of the two isoforms of CX3CL1, as both are natively present in the brain. In this study we examined each isoform's ability to regulate neuroinflammation in a mouse model of Parkinson's disease initiated by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We were able to delineate the function of both CX3CL1 isoforms by using adeno-associated virus-mediated gene therapy to selectively express synthetic variants of CX3CL1 that remain either permanently soluble or membrane bound. In the present study we injected each CX3CL1 variant or a GFP-expressing vector directly into the substantia nigra of CX3CL1(-/-) mice. Our results show that only the soluble isoform of CX3CL1 is sufficient for neuroprotection after exposure to MPTP. Specifically, we show that the soluble CX3CL1 isoform reduces impairment of motor coordination, decreases dopaminergic neuron loss, and ameliorates microglial activation and proinflammatory cytokine release resulting from MPTP exposure. Furthermore, we show that the membrane-bound isoform provides no neuroprotective capability to MPTP-induced pathologies, exhibiting similar motor coordination impairment, dopaminergic neuron loss, and inflammatory phenotypes as MPTP-treated CX3CL1(-/-) mice, which received the GFP-expressing control vector. Our results reveal that the neuroprotective capacity of CX3CL1 resides solely upon the soluble isoform in an MPTP-induced model of Parkinson's disease.