17 research outputs found
Inter-α-Inhibitor Blocks Epithelial Sodium Channel Activation and Decreases Nasal Potential Differences in ÎF508 Mice
Increased activity of lung epithelial sodium channels (ENaCs) contributes to the pathophysiology of cystic fibrosis (CF) by increasing the rate of epithelial lining fluid reabsorption. Inter-α-inhibitor (IαI), a serum protease inhibitor, may decrease ENaC activity by preventing its cleavage by serine proteases. High concentrations of IαI were detected in the bronchoalveolar lavage fluid (BALF) of children with CF and lower airway diseases. IαI decreased amiloride-sensitive (IENaC) but not cAMP-activated Clâ currents across confluent monolayers of rat ATII, and mouse nasal epithelial cells grew in primary culture by 45 and 25%, respectively. Changes in IENaC by IαI in ATII cells were accompanied by increased levels of uncleaved (immature) surface α-ENaC. IαI increased airway surface liquid depth overlying murine nasal epithelial cells to the same extent as amiloride, consistent with ENaC inhibition. Incubation of lung slices from C57BL/6, those lacking phenylalanine at position 508 (âF508), or CF transmembrane conductance regulator knockout mice with IαI for 3 hours decreased the open probability of their ENaC channels by 50%. âF508 mice had considerably higher levels the amiloride-sensitive fractions of ENaC nasal potential difference (ENaC-NPD) than wild-type littermates and only background levels of IαI in their BALF. A single intranasal instillation of IαI decreased their ENaC-NPD 24 hours later by 25%. In conclusion, we show that IαI is present in the BALF of children with CF, is an effective inhibitor of ENaC proteolysis, and decreases ENaC activity in lung epithelial cells of âF508 mice
Functional Stability of Rescued ÎF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells
The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, ÎF508, results in the production of a misfolded protein that is rapidly degraded. The mutant protein is temperature sensitive, and prior studies indicate that the low-temperatureârescued channel is poorly responsive to physiological stimuli, and is rapidly degraded from the cell surface at 37°C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxy-phenylamino)-4âČ-methyl-[4,5âČbithiazolyl-2âČ-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperatureârescued ÎF508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued ÎF508 CFTR for up to 12 hours at 37°C (P < 0.05). Using firefly luciferaseâbased reporters to investigate the mechanisms by which low temperature and corr-4a enhance rescue, we found that low-temperature treatment inhibited proteasomal function, whereas corr-4a treatment inhibited the E1-E3 ubiquitination pathway. Ussing chamber studies indicated that corr-4a increased the cAMP-mediated ÎF508 CFTR response by 61% at 6 hours (P < 0.05), but not at later time points. However, addition of the CFTR channel activator, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol, significantly augmented cAMP-stimulated currents, revealing that the biochemically detectable cell surface ÎF508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued ÎF508 CFTR was not sufficient to obtain maximal ÎF508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of ÎF508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity
Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinsonâs disease
Abstract Background Parkinsonâs disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD. Methods In vitro, we utilized microglia cultures from WT or CIITA â/â mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA â/â mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration. Results Using ICC and DQ-ovalbumin, we show that CIITA â/â microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration. Conclusion Our data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD
α-Synuclein fibrils recruit peripheral immune cells in the rat brain prior to neurodegeneration
Abstract Genetic variation in a major histocompatibility complex II (MHCII)-encoding gene (HLA-DR) increases risk for Parkinson disease (PD), and the accumulation of MHCII-expressing immune cells in the brain correlates with α-synuclein inclusions. However, the timing of MHCII-cell recruitment with respect to ongoing neurodegeneration, and the types of cells that express MHCII in the PD brain, has been difficult to understand. Recent studies show that the injection of short α-synuclein fibrils into the rat substantia nigra pars compacta (SNpc) induces progressive inclusion formation in SNpc neurons that eventually spread to spiny projection neurons in the striatum. Herein, we find that α-synuclein fibrils rapidly provoke a persistent MHCII response in the brain. In contrast, equivalent amounts of monomeric α-synuclein fail to induce MHCII or persistent microglial activation, consistent with our results in primary microglia. Flow cytometry and immunohistochemical analyses reveal that MHCII-expressing cells are composed of both resident microglia as well as cells from the periphery that include monocytes, macrophages, and lymphocytes. Over time, α-Synuclein fibril exposures in the SNpc causes both axon loss as well as monocyte recruitment in the striatum. While these monocytes in the striatum initially lack MHCII expression, α-synuclein inclusions later form in nearby spiny projection neurons and MHCII expression becomes robust. In summary, in the rat α-synuclein fibril model, peripheral immune cell recruitment occurs prior to neurodegeneration and microglia, monocytes and macrophages all contribute to MHCII expression
Ascorbate and Deferoxamine Administration after Chlorine Exposure Decrease Mortality and Lung Injury in Mice
Bakgrund: RĂ€dsla för blod och injektioner Ă€r den nĂ€st vanligaste rĂ€dslan hos barn. Barn utsĂ€tts ofta för nĂ„lrelaterade procedurer vilket kan vara en smĂ€rtsam och Ă„ngestladdad upplevelse. För mĂ„nga barn Ă€r den största rĂ€dslan vid sjukhusbesöket att fĂ„ en spruta eller att uppleva smĂ€rta. Syfte: Syftet med denna litteraturöversikt Ă€r att beskriva hur lidande och stickrĂ€dsla kan lindras hos barn vid nĂ„lrelaterade procedurer. Metod: Litteraturöversikten omfattas av 20 kvantitativa respektive 3 kvalitativa artiklar som granskades och analyserades för att sedan anvĂ€ndas i resultatet. Dessa artiklar Ă€r publicerade mellan 2006 och 2016 och sökningarna gjordes i Pubmed och Cinahl. Resultatet: Resultatet visar att smĂ€rta och Ă„ngest kan minskas hos barn under nĂ„lrelaterade procedurer med hjĂ€lp av sociala faktorer sĂ„ som förĂ€ldrar, omvĂ„rdnad och en hund. Oral och topikal smĂ€rtlindring Ă€r effektiv och distraherande metoder kan minska bĂ„de Ă„ngest och smĂ€rta. Diskussion: Ăven om distraktion har visat sig vara en effektiv metod sĂ„ Ă€r det ocksĂ„ av stor vikt att inkludera barnet i sin egen vĂ„rd. FörĂ€ldrar och sjuksköterskan tillsammans har en viktig roll att se till att barnet för en individanpassad vĂ„rd vid nĂ„lrelaterade procedurer. Slutsats: Det bör finnas olika typer av preventioner som sjuksköterskan kan tillĂ€mpa nĂ€r barn kĂ€nner smĂ€rta och Ă„ngest under nĂ„lrelaterade procedurer. Ytterligare kvalitativ och kvantitativ forskning behövs gĂ€llande barns egna önskningar vid procedurer med nĂ„lar.GodkĂ€nnande datum: 2016-10-31</p
Ascorbate and Deferoxamine Administration after Chlorine Exposure Decrease Mortality and Lung Injury in Mice
Additional file 1: of ĂÄ -Synuclein fibrils recruit peripheral immune cells in the rat brain prior to neurodegeneration
Supplemental figures. (PDF 31368 kb