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INTRODUCTION: The understanding of the pathological events in Alzheimer's disease (AD) has advanced dramatically, but the successful translation from rodent models into efficient human therapies is still problematic. METHODS: To examine how tau pathology can develop in the primate brain, we injected 12 macaques with a dual tau mutation (P301L/S320F) into the entorhinal cortex (ERC). An investigation was performed using high-resolution microscopy, magnetic resonance imaging (MRI), positron emission tomography (PET), and fluid biomarkers to determine the temporal progression of the pathology 3 and 6 months after the injection. RESULTS: Using quantitative microscopy targeting markers for neurodegeneration and neuroinflammation, as well as fluid and imaging biomarkers, we detailed the progression of misfolded tau spreading and the consequential inflammatory response induced by glial cells. DISCUSSION: By combining the analysis of several in vivo biomarkers with extensive brain microscopy analysis, we described the initial steps of misfolded tau spreading and neuroinflammation in a monkey model highly translatable to AD patients. HIGHLIGHTS: Dual tau mutation delivery in the entorhinal cortex induces progressive tau pathology in rhesus macaques. Exogenous human 4R-tau coaptates monkey 3R-tau during transneuronal spread, in a prion-like manner. Neuroinflammatory response is coordinated by microglia and astrocytes in response to tau pathology, with microglia targeting early tau pathology, while astrocytes engaged later in the progression, coincident with neuronal death. Monthly collection of CSF and plasma revealed a profile of changes in several AD core biomarkers, reflective of neurodegeneration and neuroinflammation as early as 1 month after injection.
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Doença de Alzheimer , Modelos Animais de Doenças , Progressão da Doença , Macaca mulatta , Tomografia por Emissão de Pósitrons , Proteínas tau , Animais , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Imageamento por Ressonância Magnética , Doenças Neuroinflamatórias/patologia , Córtex Entorrinal/patologia , Córtex Entorrinal/metabolismo , Biomarcadores , Mutação , Encéfalo/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , MasculinoRESUMO
CD4+ T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-Seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4+ T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4+ T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4+ T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL757 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4+ T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4+ T cells in CNS immune surveillance, and their decline during chronic SIV highlights their responsiveness to neuroinflammation.
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Encéfalo , Linfócitos T CD4-Positivos , Macaca mulatta , Receptores CCR7 , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T CD4-Positivos/imunologia , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores CCR7/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Encéfalo/patologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/patologia , Vigilância ImunológicaRESUMO
Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS)-known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Acute infection led to marked imbalance in the CNS CD4/CD8 ratio which persisted into the chronic phase. These observations underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our findings offer insights for potential T cell-focused interventions while underscoring the challenges in eradicating HIV from the CNS, particularly in the context of sub-optimal ART.
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Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Humanos , Linfócitos T CD4-Positivos , Vírus da Imunodeficiência Símia/fisiologia , Macaca mulatta , Sistema Nervoso Central , Carga ViralRESUMO
CD4 T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4 T cells resembling lymph node central memory (T CM ) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of T CM . Brain CCR7+ CD4 T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside other CNS border tissues. Sequestering T CM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4 T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL57 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4 T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4 T cells in CNS immune surveillance and their decline during chronic SIV-induced neuroinflammation highlights their responsiveness to neuroinflammatory processes. In Brief: Utilizing single-cell and spatial transcriptomics on adult rhesus brain, we uncover a unique CCR7+ CD4 T cell subset resembling central memory T cells (T CM ) within brain and border tissues, including skull bone marrow. Our findings show decreased frequencies of this subset during SIV- induced chronic neuroinflammation, emphasizing responsiveness of CCR7+ CD4 T cells to CNS disruptions. Highlights: CCR7+ CD4 T cells survey border and parenchymal CNS compartments during homeostasis; reduced presence of CCR7+ CD4 T cells in cerebrospinal fluid leads to immune activation, implying a role in neuroimmune homeostasis. CNS CCR7+ CD4 T cells exhibit phenotypic and functional features of central memory T cells (T CM ) including production of interleukin 2 and the capacity for rapid recall proliferation. Furthermore, CCR7+ CD4 T cells reside in the skull bone marrow. CCR7+ CD4 T cells are markedly decreased within the brain parenchyma during chronic viral neuroinflammation.
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Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS) - known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Despite viral suppression with ART, acute infection led to significant depletion of CNS CD4 T cells, persisting into the chronic phase. These findings underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our results offer insights for potential T cell-focused interventions while also underscoring the challenges in eradicating HIV from the CNS, even with effective ART.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), can induce a plethora of neurological complications in some patients. However, it is still under debate whether SARS-CoV-2 directly infects the brain or whether CNS sequelae result from systemic inflammatory responses triggered in the periphery. By using high-resolution microscopy, we investigated whether SARS-CoV-2 reaches the brain and how viral neurotropism can be modulated by aging in a non-human primate model of COVID-19. Seven days after infection, SARS-CoV-2 was detected in the olfactory cortex and interconnected regions and was accompanied by robust neuroinflammation and neuronal damage exacerbated in aged, diabetic animals. Our study provides an initial framework for identifying the molecular and cellular mechanisms underlying SARS-CoV-2 neurological complications, which will be essential to reducing both the short- and long-term burden of COVID-19.
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COVID-19 , Doenças do Sistema Nervoso , Animais , SARS-CoV-2 , Doenças Neuroinflamatórias , Neurônios , PrimatasRESUMO
BACKGROUND: Immunosurveillance of the central nervous system (CNS) is vital to resolve infection and injury. However, immune activation within the CNS in the setting of chronic viral infections, such as HIV-1, is strongly linked to progressive neurodegeneration and cognitive decline. Establishment of HIV-1 in the CNS early following infection underscores the need to delineate features of acute CNS immune activation, as these early inflammatory events may mediate neurodegenerative processes. Here, we focused on elucidating molecular programs of neuroinflammation in brain regions based on vulnerability to neuroAIDS and/or neurocognitive decline. To this end, we assessed transcriptional profiles within the subcortical white matter of the pre-frontal cortex (PFCw), as well as synapse dense regions from hippocampus, superior temporal cortex, and caudate nucleus, in rhesus macaques following infection with Simian/Human Immunodeficiency Virus (SHIV.C.CH505). METHODS: We performed RNA extraction and sequenced RNA isolated from 3 mm brain punches. Viral RNA was quantified in the brain and cerebrospinal fluid by RT-qPCR assays targeting SIV Gag. Neuroinflammation was assessed by flow cytometry and multiplex ELISA assays. RESULTS: RNA sequencing and flow cytometry data demonstrated immune surveillance of the rhesus CNS by innate and adaptive immune cells during homeostasis. Following SHIV infection, viral entry and integration within multiple brain regions demonstrated vulnerabilities of key cognitive and motor function brain regions to HIV-1 during the acute phase of infection. SHIV-induced transcriptional alterations were concentrated to the PFCw and STS with upregulation of gene expression pathways controlling innate and T-cell inflammatory responses. Within the PFCw, gene modules regulating microglial activation and T cell differentiation were induced at 28 days post-SHIV infection, with evidence for stimulation of immune effector programs characteristic of neuroinflammation. Furthermore, enrichment of pathways regulating mitochondrial respiratory capacity, synapse assembly, and oxidative and endoplasmic reticulum stress were observed. These acute neuroinflammatory features were substantiated by increased influx of activated T cells into the CNS. CONCLUSIONS: Our data show pervasive immune surveillance of the rhesus CNS at homeostasis and reveal perturbations of important immune, neuronal, and synaptic pathways within key anatomic regions controlling cognition and motor function during acute HIV infection. These findings provide a valuable framework to understand early molecular features of HIV associated neurodegeneration.
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Infecções por HIV , HIV-1 , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Substância Branca , Animais , Lobo Frontal , HIV-1/genética , Humanos , Macaca mulatta/genética , RNA Viral , Carga ViralRESUMO
INTRODUCTION: Alzheimer's disease (AD) is a devastating condition with no effective treatments, with promising findings in rodents failing to translate into successful therapies for patients. METHODS: Targeting the vulnerable entorhinal cortex (ERC), rhesus monkeys received two injections of an adeno-associated virus expressing a double tau mutation (AAV-P301L/S320F) in the left hemisphere, and control AAV-green fluorescent protein in the right ERC. Noninjected aged-matched monkeys served as additional controls. RESULTS: Within 3 months we observed evidence of misfolded tau propagation, similar to what is hypothesized to occur in humans. Viral delivery of human 4R-tau also coaptates monkey 3R-tau via permissive templating. Tau spreading is accompanied by robust neuroinflammatory response driven by TREM2+ microglia, with biomarkers of inflammation and neuronal loss in the cerebrospinal fluid and plasma. DISCUSSION: These results highlight the initial stages of tau seeding and propagation in a primate model, a more powerful translational approach for the development of new therapies for AD.
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Doença de Alzheimer/patologia , Modelos Animais de Doenças , Macaca mulatta/metabolismo , Proteínas tau/líquido cefalorraquidiano , Idoso , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Córtex Entorrinal/patologia , Feminino , Humanos , Microglia/metabolismo , Mutação/genéticaRESUMO
As the average age of the population continues to rise, the number of individuals affected with age-related cognitive decline and Alzheimer's disease (AD) has increased and is projected to cost more than $290 billion in the United States in 2019. Despite significant investment in research over the last decades, there is no effective treatment to prevent or delay AD progression. There is a translational gap in AD research, with promising drugs based on work in rodent models failing in clinical trials. Aging is the leading risk factor for developing AD and understanding neurobiological changes that affect synaptic integrity with aging will help clarify why the aged brain is vulnerable to AD. We describe here the development of a rhesus monkey model of AD using soluble oligomers of the amyloid beta (Aß) peptide (AßOs). AßOs infused into the monkey brain target a specific population of spines in the prefrontal cortex, induce neuroinflammation, and increase AD biomarkers in the cerebrospinal fluid to similar levels observed in patients with AD. Importantly, AßOs lead to similar dendritic spine loss to that observed in normal aging in monkeys, but so far without detection of amyloid plaques or tau pathology. Understanding the basis of synaptic impairment is the most effective route to early intervention and prevention or postponement of age-related cognitive decline and transition to AD. These initial findings support the use of monkeys as a platform to understand age-related vulnerabilities of the primate brain and may help develop effective disease-modifying therapies for treatment of AD and related dementias.
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RATIONALE: Cystic fibrosis (CF) patients are known to produce cyanide (CN-) although challenges exist in determinations of total levels, the precise bioactive levels, and specificity of its production by CF microflora, especially P. aeruginosa. Our objective was to measure total CN- levels in CF sputa by a simple and novel technique in P. aeruginosa positive and negative adult patients, to review respiratory tract (RT) mechanisms for the production and degradation of CN-, and to interrogate sputa for post-translational protein modification by CN- metabolites. METHODS: Sputa CN- concentrations were determined by using a commercially available CN- electrode, measuring levels before and after addition of cobinamide, a compound with extremely high affinity for CN-. Detection of protein carbamoylation was measured by Western blot. MEASUREMENTS AND MAIN RESULTS: The commercial CN- electrode was found to overestimate CN- levels in CF sputum in a highly variable manner; cobinamide addition rectified this analytical issue. Although P. aeruginosa positive patients tended to have higher total CN- values, no significant differences in CN- levels were found between positive and negative sputa. The inflammatory oxidant hypochlorous acid (HOCl) was shown to rapidly decompose CN-, forming cyanogen chloride (CNCl) and the carbamoylating species cyanate (NCO-). Carbamoylated proteins were found in CF sputa, analogous to reported findings in asthma. CONCLUSIONS: Our studies indicate that CN- is a transient species in the inflamed CF airway due to multiple biosynthetic and metabolic processes. Stable metabolites of CN-, such as cyanate, or carbamoylated proteins, may be suitable biomarkers of overall CN- production in CF airways.
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Cianetos/análise , Fibrose Cística/metabolismo , Técnicas Eletroquímicas , Ácido Hipocloroso/química , Processamento de Proteína Pós-Traducional , Escarro/química , Adulto , Cobamidas/química , Cianetos/metabolismo , Fibrose Cística/diagnóstico , Fibrose Cística/microbiologia , Eletrodos , Feminino , Humanos , Ácido Hipocloroso/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Oxirredução , Carbamilação de Proteínas , Pseudomonas aeruginosa/metabolismo , Escarro/microbiologiaRESUMO
Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 µM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.
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Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Farnesiltranstransferase/antagonistas & inibidores , Inflamação/tratamento farmacológico , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Proteínas ras/metabolismo , Animais , Asma/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Hiper-Reatividade Brônquica/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Eosinófilos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Farnesiltranstransferase/metabolismo , Humanos , Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.
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Autofagia , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/patologia , Pulmão/efeitos dos fármacos , Fator de Crescimento Transformador beta1/administração & dosagem , Resposta a Proteínas não Dobradas , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Transdução de SinaisRESUMO
Systemic treatment with statins mitigates allergic airway inflammation, TH2 cytokine production, epithelial mucus production, and airway hyperreactivity (AHR) in murine models of asthma. We hypothesized that pravastatin delivered intratracheally would be quantifiable in lung tissues using mass spectrometry, achieve high drug concentrations in the lung with minimal systemic absorption, and mitigate airway inflammation and structural changes induced by ovalbumin. Male BALB/c mice were sensitized to ovalbumin (OVA) over 4 weeks, then exposed to 1% OVA aerosol or filtered air (FA) over 2 weeks. Mice received intratracheal instillations of pravastatin before and after each OVA exposure (30 mg/kg). Ultra performance liquid chromatography - mass spectrometry was used to quantify plasma, lung, and bronchoalveolar lavage fluid (BALF) pravastatin concentration. Pravastatin was quantifiable in mouse plasma, lung tissue, and BALF (BALF > lung > plasma for OVA and FA groups). At these concentrations pravastatin inhibited airway goblet cell hyperplasia/metaplasia, and reduced BALF levels of cytokines TNFα and KC, but did not reduce BALF total leukocyte or eosinophil cell counts. While pravastatin did not mitigate AHR, it did inhibit airway hypersensitivity (AHS). In this proof-of-principle study, using novel mass spectrometry methods we show that pravastatin is quantifiable in tissues, achieves high levels in mouse lungs with minimal systemic absorption, and mitigates some pathological features of allergic asthma. Inhaled pravastatin may be beneficial for the treatment of asthma by having direct airway effects independent of a potent anti-inflammatory effect. Statins with greater lipophilicity may achieve better anti-inflammatory effects warranting further research.
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Myeloperoxidase (MPO) produces hypohalous acids as a key component of the innate immune response; however, release of these acids extracellularly results in inflammatory cell and tissue damage. The two-step, one-pot Davis-Beirut reaction was used to synthesize a library of 2H-indazoles and 1H-indazolones as putative inhibitors of MPO. A structure-activity relationship study was undertaken wherein compounds were evaluated utilizing taurine-chloramine and MPO-mediated H2O2 consumption assays. Docking studies as well as toxicophore and Lipinski analyses were performed. Fourteen compounds were found to be potent inhibitors with IC50 values <1µM, suggesting these compounds could be considered as potential modulators of pro-oxidative tissue injury pertubated by the inflammatory MPO/H2O2/HOCl/HOBr system.
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Indazóis/química , Peroxidase/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Cloraminas/química , Cloraminas/metabolismo , Humanos , Indazóis/metabolismo , Simulação de Acoplamento Molecular , Peroxidase/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Taurina/química , Taurina/metabolismoRESUMO
The transcriptome of ataxic muscles from alpha-tocopherol transfer protein deficient (ATTP-KO), 23-month old, mice was compared with that of their normal littermates. Genes encoding sarcolipin (sln) and ubiquitin carboxyl-terminal hydrolase (uchl1) were over-expressed (> or =10-fold) in ataxic muscles. SLN is a 3.2 kDa membrane protein that binds to sarcoplasmic reticulum calcium ATPase, regulates Ca(+ +) transport and muscle relaxation-contraction cycles. UCHL1 is a 24.8 kDa member of proteosome proteins; it is over-expressed in myofibrillar myopathy and is associated with neurodegenerative diseases. Furthermore, six additional transcripts, three encoding thin-filament proteins and three encoding Ca(+ +) sensing proteins that participate in contraction-relaxation cycle, and eight transcripts that encode members of lysosomal proteins were also over-expressed in ataxic muscles. These observations suggest that chronic alpha-tocopherol (AT) deficiency activates critical genes of muscle contractility and protein degradation pathways, simultaneously. The magnitude of induction of sln and uchl1 was lower in asymptomatic, 8-month old, ATTP-KO mice and in 8-month old mice fed an AT-depleted diet. These studies suggest sln and uchl1 genes as novel targets of AT deficiency and may offer molecular correlates of well documented descriptions of neuromuscular dysfunctions in AT-deficient rodents. Since the neuromuscular deficits of ATTP-KO mice appear to be similar to those of patients with ATTP mutations, it is suggested that over-expression of sln and uchl1 may also contribute to AT-sensitive ataxia in humans.