RESUMO
INTRODUCTION: High levels of serum food-specific IgG4 (sIgG4) have been reported in patients with EoE. The objective of this study was to examine whether serum sIgG4 levels to foods and aeroallergens are higher in EoE patients than allergic controls and to investigate the association between sIgG4 and EoE clinical characteristics. METHODS: This was a case-control study nested in a prospective EoE Cohort. EoE cases were defined per consensus guidelines, and controls were individuals with symptoms who were confirmed to be EoE-negative on upper endoscopy. Demographic and clinical information was prospectively collected. Serum IgE and sIgG4 were measured to foods and aeroallergens by ImmunoCAP. Mean levels of sIgG4 were compared between cases and controls, and logistic regression models were used to examine predictors of elevated milk sIgG4 levels. RESULTS: The analysis included 123 individuals (EoE n = 93, control n = 30) with a similar distribution of allergic disease between EoE patients and controls (86% vs. 93%; p = .30). EoE patients had significantly higher sIgG4 levels to all allergens evaluated, with the exception of birch (p = .24). Milk sIgG4 levels were independently associated with milk consumption (OR 4.95; p = .01) and the presence of sIgE to milk (OR 4.23; p = .008). CONCLUSION: Serum sIgG4 levels to food and aeroallergen proteins were higher in patients with EoE than non-EoE controls, and higher levels of milk sIgG4 were independently associated with milk consumption and the presence of sIgE to milk proteins. Whether sIgG4 plays a pathogenic role in EoE or could be used as an EoE biomarker remains unknown and warrants further study.
Assuntos
Esofagite Eosinofílica , Humanos , Animais , Estudos Prospectivos , Imunoglobulina G , Estudos de Casos e Controles , Imunoglobulina E , Alérgenos , LeiteRESUMO
BACKGROUND: Recent studies have shown deposition of immunoglobulin G4 (IgG4) and food proteins in the esophageal mucosa of eosinophilic esophagitis (EoE) patients. Our aims were to assess whether co-localization of IgG4 and major cow's milk proteins (CMPs) was associated with EoE disease activity and to investigate the proteins enriched in proximity to IgG4 deposits. METHODS: This study included adult subjects with EoE (n = 13) and non-EoE controls (n = 5). Esophageal biopsies were immunofluorescence stained for IgG4 and CMPs. Co-localization in paired samples from active disease and remission was assessed and compared to controls. The proteome surrounding IgG4 deposits was evaluated by the novel technique, AutoSTOMP. IgG4-food protein interactions were confirmed with co-immunoprecipitation and mass spectrometry. RESULTS: IgG4-CMP co-localization was higher in the active EoE group compared to paired remission samples (Bos d 4, p = .02; Bos d 5, p = .002; Bos d 8, p = .002). Co-localization was also significantly higher in the active EoE group compared to non-EoE controls (Bos d 4, p = .0013; Bos d 5, p = .0007; Bos d 8, p = .0013). AutoSTOMP identified eosinophil-derived proteins (PRG 2 and 3, EPX, RNASE3) and calpain-14 in IgG4-enriched areas. Co-immunoprecipitation and mass spectrometry confirmed IgG4 binding to multiple food allergens. CONCLUSION: These findings further contribute to the understanding of the interaction of IgG4 with food antigens as it relates to EoE disease activity. These data strongly suggest the immune complex formation of IgG4 and major cow's milk proteins. These immune complexes may have a potential role in the pathophysiology of EoE by contributing to eosinophil activation and disease progression.
Assuntos
Esofagite Eosinofílica , Adulto , Feminino , Animais , Bovinos , Humanos , Esofagite Eosinofílica/patologia , Complexo Antígeno-Anticorpo , Imunoglobulina G , Alérgenos , Proteínas do LeiteRESUMO
Tissue microenvironment properties like blood flow, extracellular matrix, or proximity to immune-infiltrate are important regulators of cell biology. However, methods to study regional protein expression in the native tissue environment are limited. To address this need, we developed a novel approach to visualize, purify, and measure proteins in situ using automated spatially targeted optical microproteomics (AutoSTOMP). Here, we report custom codes to specify regions of heterogeneity in a tissue section and UV-biotinylate proteins within those regions. We have developed liquid chromatography-mass spectrometry (LC-MS)/MS-compatible biochemistry to purify those proteins and label-free quantification methodology to determine protein enrichment in target cell types or structures relative to nontarget regions in the same sample. These tools were applied to (a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and (b) characterize inflammatory proteins enriched in IgG4+ lesions in human esophageal tissues. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools and sample availability are limited.
Assuntos
Proteínas , Proteômica , Animais , Cromatografia Líquida , Espectrometria de Massas , RatosRESUMO
PURPOSE OF REVIEW: Progestogen hypersensitivity (PH) is a rare disorder which usually occurs in women of childbearing age with symptoms ranging from urticaria with or without angioedema, multiple organ involvement consistent with allergic anaphylaxis, to a spectrum of other non-evanescent skin eruptions. In this review, we present a clinical vignette of PH and discuss the clinical presentation and proposed pathomechanisms, diagnosis, and treatment of PH. RECENT FINDINGS: The hypersensitivity symptoms are associated with exogenous progestin exposure (e.g., contraceptive medicines, in vitro fertilization therapy) or endogenous progesterone from progesterone surges during the luteal phase of the menstrual cycle and pregnancy. Recognition of this condition can be challenging to the clinician due to its heterogeneous clinical presentation. It has been recently proposed to use the new term "progestogen hypersensitivity" to replace "autoimmune progesterone dermatitis" due to the lack of evidence supporting an autoimmune mechanism for this disorder. In addition, diagnostic and treatment algorithms are now available that can lead to successful management of this condition. More new developments of Progesterone desensitization protocols are now available which appear to be the safest and most effective long-term treatment option for PH. With the extensive use of oral contraceptives and increased use of supra-physiologic doses of progesterone to support pregnancy in in vitro fertilization, there is likely to be a higher prevalence of PH in the future than currently recognized. Therefore, the allergist-immunologist will be required to collaborate with gynecologists and reproductive endocrinologists to diagnose and treat this condition.
Assuntos
Doenças Autoimunes/diagnóstico , Dermatite/diagnóstico , Dessensibilização Imunológica/métodos , Progesterona/efeitos adversos , Progestinas/efeitos adversos , Adulto , Doenças Autoimunes/patologia , Dermatite/patologia , Feminino , HumanosRESUMO
BACKGROUND: Diagnosis of VCD is complicated by its symptom similarities to asthma. Although clinical history, spirometry, and fiberoptic nasolaryngoscopy are used for VCD diagnosis, videostroboscopy is considered the gold standard. However, little is know about patient characteristics that might suggest a VCD diagnosis is more likely. OBJECTIVE: To identify clinical characteristics of patients suspected of having VCD that would increase the likelihood of an accurate diagnosis before videostroboscopy. METHODS: Records of 55 patients were reviewed for a cross-sectional, retrospective study. Individuals selected were suspected of having VCD because of poor clinical response to asthma medications, absence of objective criteria for diagnosis of asthma (eg, normal forced expiratory volume in 1 second without reversibility, normal exhaled nitric oxide, equivocal methacholine challenge test), or both. We used χ2 analyses to determine significant univariate associations of various patient characteristics. Multivariate regression analysis was then performed using those variables identified as being significant predictors by univariate analysis. RESULTS: A significant association between VCD and age and between VCD and shortness of breath (SOB) was found. Further analysis revealed that at ages younger than 35 years, with every 5-year decrement in age, patients suspected of having VCD in which SOB is the presenting symptom are more likely to have a positive VCD diagnosis by a factor of 1.3. CONCLUSION: Clinical presentation of younger patients with SOB in conjunction with lack of objective criteria for an asthma diagnosis, poor response to asthma medications, or both is highly predictive of VCD and should prompt an objective stroboscopic evaluation to confirm the diagnosis.
Assuntos
Disfunção da Prega Vocal/diagnóstico , Adolescente , Adulto , Criança , Tosse/diagnóstico , Estudos Transversais , Dispneia/diagnóstico , Feminino , Rouquidão/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Estroboscopia , Adulto JovemAssuntos
Complemento C2/genética , Síndromes de Imunodeficiência/diagnóstico , Deleção de Sequência/genética , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/fisiologia , Apneia , Feminino , Febre , Homozigoto , Humanos , Síndromes de Imunodeficiência/genética , Lactente , Convulsões , Infecções Estreptocócicas/genéticaRESUMO
Introduction: Depression is strongly associated with Alzheimer's disease (AD). Antidepressants are commonly used in patients before and after their diagnosis of AD. To date, the relationship between antidepressants and AD remains unclear. Methods: In our study, we administered sertraline or paroxetine to wild type (WT) and APPswe/PSEN1dE9 (APP/PSEN1) transgenic mouse models for up to 12 months. We quantified the drug concentrations using LC-MS/MS analysis and measured serum serotonin level using an ELISA assay. Additionally, we evaluated the amyloid burdens through thioflavin-S and Congo red stainings, and recognition memory using the novel object recognition test. Results: Our findings revealed that mice treated with paroxetine exhibited a significantly higher level of weight gain compared to the control group and increased mortality in APP/PSEN1 mice. After 12 months of antidepressant treatment, the sertraline level was measured at 289.8 ng/g for cerebellum, while the paroxetine level was 792.9 ng/g for cerebellum. Sertraline significantly increased thioflavin-S and Congo red depositions, along with gliosis, in both isocortex and hippocampus of APP/PSEN1 mice compared to the control group. Both antidepressants also led to a decreased recognition index in APP/PSEN1 mice. Conclusion: These findings suggest a potential role of sertraline in AD pathogenesis, emphasizing the need to reassess the use of these antidepressants in patients with AD.
RESUMO
It has become increasingly clear that inflammatory processes play a significant role in the pathophysiology of Alzheimer's disease (AD). Neuroinflammation is characterized by the activation of astrocytes and microglia and the release of proinflammatory cytokines and chemokines. Vascular inflammation, mediated largely by the products of endothelial activation, is accompanied by the production and the release of a host of inflammatory factors which contribute to vascular, immune, and neuronal dysfunction. The complex interaction of these processes is still only imperfectly understood, yet as the mechanisms continue to be elucidated, targets for intervention are revealed. Although many of the studies to date on therapeutic or preventative strategies for AD have been narrowly focused on single target therapies, there is accumulating evidence to suggest that the most successful treatment strategy will likely incorporate a sequential, multifactorial approach, addressing direct neuronal support, general cardiovascular health, and interruption of deleterious inflammatory pathways.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Mediadores da Inflamação/fisiologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , Microglia/metabolismo , Microglia/patologiaRESUMO
Antibodies of the IgG4 isotype are strongly associated with allergic disease but have several properties such as not precipitating with allergens, not activating complement and poor binding to Fcγ receptors that argue against a pro-inflammatory role. In keeping with that, IgG4 antibodies are a striking feature of the response to immunotherapy. In two naturally occurring situations IgG4 antibodies are common with low or absent IgE antibodies. The first example is children raised in a house with a cat and the second is eosinophilic esophagitis (EoE). In many population-based cohorts, the ownership of a cat in early childhood is associated with a decreased prevalence of a cat allergy at age 10. The second example (i.e., EoE) is a novel form of food allergy that is not mediated by IgE and is related to consuming cow's milk or wheat. In EoE, patients have IgG4 to milk proteins in high > 10 µg/mL or very high > 100 µg/mL titers. Enigmatically these patients are found to have deposits of IgG4 in the wall of their inflamed esophagus. The factors that have given rise to EoE remain unclear; however, changes in food processing over the past 50 years, particularly ultra-heat treatment and the high pressure homogenization of milk, represent a logical hypothesis.
RESUMO
BACKGROUND: The enzyme cytosolic phospholipase A2 alpha (cPLA2alpha) has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA2alpha enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA2alpha in early ischemic cerebral injury. METHODS: Middle cerebral artery occlusion (MCAO) was performed on cPLA2alpha+/+ and cPLA2alpha-/- mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA2alpha, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE2 content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion. RESULTS: Neuronal cPLA2alpha protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE2 concentration were greater in cPLA2alpha+/+ compared to cPLA2alpha-/- ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA2alpha+/+ than in cPLA2alpha-/- brains (+/+:2.2+/-0.3 fold vs. -/-:1.7+/-0.4 fold increase; P<0.01). The increase in reactive oxygen species following 2 hours of ischemia was also significantly greater in the cPLA2alpha+/+ ischemic core than in cPLA2alpha-/- (+/+:7.12+/-1.2 fold vs. -/-:3.1+/-1.4 fold; P<0.01). After 6 hours of reperfusion ischemic cortex of cPLA2alpha+/+, but not cPLA2alpha-/-, had disruption of neuron morphology and decreased PGE2 content. Phosphorylation of the MAPKs-p38, ERK 1/2, and MEK 1/2-was significantly greater in cPLA2a+/+ than in cPLA2alpha-/- ischemic cortex 6 hours after reperfusion. CONCLUSIONS: These results indicate that cPLA2alpha modulates the earliest molecular and injury responses after cerebral ischemia and have implications for the potential clinical use of cPLA2alpha inhibitors.
Assuntos
Isquemia Encefálica/fisiopatologia , Ciclo-Oxigenase 2/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/fisiopatologia , Animais , Isquemia Encefálica/metabolismo , Dinoprostona/metabolismo , Feminino , Fosfolipases A2 do Grupo IV/genética , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , FosforilaçãoRESUMO
Food allergy is often understood as an IgE-mediated hypersensitivity, characterized by allergic symptoms which occur "immediately" after the ingestion of a relevant food allergen. Increasingly, however, other food-related immune-mediated disorders are recognized in which symptoms can have a delayed onset and IgE does not play a central role. One of the described examples of the latter is eosinophilic esophagitis (EoE) - a disease defined pathologically by local eosinophilic inflammation in the esophagus in the setting of symptoms of esophageal dysfunction. The evidence that EoE is a food-mediated allergic disease includes i) almost all patients respond to an elemental diet and many respond to a diet in which dairy, wheat, eggs and/or soy are eliminated, ii) the presence of food-specific IgE and Th2 cells are consistent with a loss of tolerance to trigger foods and iii) many EoE patients have concomitant IgE-mediated food allergy and other allergic co-morbidities. This narrative review focuses on the hypothesis that EoE is a form of chronic food allergy. The goal is to describe similarities and differences in EoE and IgE-mediated food allergy, and to consider ways that these two increasingly common forms of food allergy are related to each other.
RESUMO
Allergic rhinitis (AR), most presentations of nasal polyposis (NP), and many presentations of chronic rhinosinusitis are type 2high disorders characterized by expression of interleukin (IL)-4, IL-5, and IL-13. Neutralization of IgE with anti-IgE (omalizumab) has proven efficacy in AR. Similarly, in addition to anti-IgE, blockade of IL-5/IL-5 (mepolizumab, reslizumab, benralizumab) and dual blockade of IL-4 and IL-13 with anti-IL-4R (dupilumab) have demonstrated efficacy in NP. However, these agents are expensive and future studies are essential to evaluate cost effectiveness in comparison with current medical and surgical therapies. This article reviews biologics as potential interventions in AR, chronic rhinosinusitis, and NP.
Assuntos
Produtos Biológicos/uso terapêutico , Pólipos Nasais/tratamento farmacológico , Rinite Alérgica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sinusite/tratamento farmacológico , Produtos Biológicos/economia , Produtos Biológicos/farmacologia , Doença Crônica/tratamento farmacológico , Doença Crônica/economia , Análise Custo-Benefício , Custos de Medicamentos , Humanos , Imunoglobulina E/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Subunidade alfa de Receptor de Interleucina-4/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Interleucina-5/antagonistas & inibidores , Interleucina-5/metabolismo , Pólipos Nasais/diagnóstico , Pólipos Nasais/economia , Pólipos Nasais/imunologia , Rinite Alérgica/diagnóstico , Rinite Alérgica/economia , Rinite Alérgica/imunologia , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Sinusite/diagnóstico , Sinusite/economia , Sinusite/imunologia , Resultado do TratamentoRESUMO
The galactose-α-1,3-galactose (α-Gal) syndrome has many novel features that are relevant to diagnosis and management. In most cases, the diagnosis can be made on a history of delayed allergic reactions to mammalian meat and the blood test for IgE to the oligosaccharide α-Gal. In general, the diagnosis also dictates the primary treatment, that is, avoiding mammalian meat and also dairy in some cases. In the United States, the lone star tick is the primary cause of this disease, but different ticks are responsible in other countries. Blood levels of IgE to α-Gal often drop in patients who avoid recurrent tick bites, but the rate of decline is variable. Similarly, the delay before reactions is variable and the severity of the allergic reactions is not predicted by the delay or the titer of specific IgE. Some mammalian-derived products such as heart valves, gelatin-based plasma expanders, and pancreatic enzymes are relevant to only select patient groups. A minority of cases may benefit from avoiding a wide range of products that are prepared with mammalian-derived constituents, such as gelatin. This review focuses on the nature of the syndrome, common challenges in diagnosis and management, and also gaps in our current knowledge that would benefit from additional investigation.
Assuntos
Hipersensibilidade Alimentar , Picadas de Carrapatos , Alérgenos , Animais , Hipersensibilidade Alimentar/diagnóstico , Hipersensibilidade Alimentar/terapia , Galactose , Humanos , Imunoglobulina ERESUMO
Ischemic preconditioning (IPC) protects brain against ischemic injury by activating specific mechanisms. Our goal was to determine if the inducible heme oxygenase 1 (HO1) is required for such protection. IPC before transient or permanent ischemia reduced cortical infarct volumes by 57.4% and 33.9%, respectively at 48 h in wildtype adult mice. Interestingly, IPC failed to protect the HO1 gene deleted mice against permanent ischemic brain injury. IPC also resulted in a significant increase in HO1 protein levels in the brain and correlated with reduced neurological deficits after permanent and transient brain ischemia. Our study demonstrates that neuroprotective effects of IPC are at least partially mediated via HO1. Elucidating the physiological/cellular role by which HO1 is protective against brain ischemia may aid the development of selective drugs to treat stroke and its associated neurological disorders.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Heme Oxigenase-1/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/prevenção & controle , Precondicionamento Isquêmico , Animais , Western Blotting , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/patologia , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/genética , Infarto da Artéria Cerebral Média/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de DoençaRESUMO
Prostanoids can suppress vascular smooth muscle cell (VSMC) proliferation, but the mechanism through which this is mediated has not been identified. In this study, we show rat aortic VSMCs to express the EP1, EP2, EP3, EP4, and IP receptors. The EP4 receptor-specific agonist, 11-deoxy-PGE1, induced a time-dependent phosphorylation of protein kinase C and extracellular signal-regulated kinase (ERK) 1/2 in serum-depleted (0.1%) VSMCs, whereas the EP2 receptor agonist, butaprost, was without effect. PGI2 or iloprost at the IP receptor inhibited basal ERK phosphorylation with IC50 values of 10 nmol/L. Iloprost also attenuated the sustained activation of ERK induced by endothelin-1 or basic fibroblast growth factor (bFGF). Endothelin-1 or bFGF significantly increased the number of VSMCs counted 24 hours later compared with basal, and both responses were blocked by the MEK inhibitor, U0126, or iloprost. Under basal conditions, U0126 or iloprost reduced the number of viable cells and increased caspase-3 activity, which could be reversed by coapplication with endothelin-1, bFGF, or the adenylate cyclase inhibitor, SQ22536. Endothelin-1, bFGF, or SQ22536 prevented the depression to below basal levels of ERK phosphorylation induced by iloprost. Forskolin activated caspase-3 and attenuated basal ERK phosphorylation, which were prevented by SQ22536, endothelin-1, or bFGF. These data suggest that iloprost induces apoptosis via a cAMP-mediated suppression of ERK activity. In turn, this apoptotic response can be blocked by a mitogenic stimulus that re-establishes ERK activity back to basal levels, but at the expense of any concomitant proliferative activity. However, ERK stimulation by a selective EP4 receptor agonist, suggests that prostanoids may have diverse and complex roles in VSMC physiology.
Assuntos
Alprostadil/análogos & derivados , Apoptose/efeitos dos fármacos , Endotelina-1/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Iloprosta/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Alprostadil/farmacologia , Animais , Butadienos/farmacologia , Caspase 3 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Colforsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Epoprostenol/farmacologia , MAP Quinase Quinase Quinases , Microscopia Confocal , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/enzimologia , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Receptores de Prostaglandina E/efeitos dos fármacos , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4RESUMO
BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) can sufficiently and independently induce pathophysiological angiogenesis. However, the treatment strategies have mostly been unsuccessful. The present study is the first to evaluate the possible targeting of downstream signals for the inhibition of HGF/SF-induced angiogenesis. METHODS AND RESULTS: In a multichannel scratch assay with human endothelial cells (ECs), HGF/SF induced a strong and prolonged activation of MAPK and cell proliferation that was inhibited by PD98059 and LY294002/wortmannin, selective inhibitors of MAPK and PI3K signaling modules, respectively. Western blotting demonstrated a temporal relation between the activation of the two pathways. Chemical inhibition of the PI3K and MAPK signals inhibited HGF/SF-induced chemoinvasion of ECs in vitro and blocked the HGF/SF-induced neovascularization into a polymer scaffold in vivo, as quantified by vessel counts and the clearance of radioactive 133Xe. CONCLUSIONS: These data indicate that MEK and PI3K inhibitors represent a promising approach to the clinical management of pathological conditions characterized by overt HGF/SF-induced angiogenesis.
Assuntos
Inibidores Enzimáticos/farmacologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases , Androstadienos/farmacologia , Animais , Bioensaio , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Colágeno , Combinação de Medicamentos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Flavonoides/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Laminina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteoglicanas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , WortmaninaRESUMO
Although acute ischemic stroke has high mortality and morbidity rate but yet still has very limited treatment. In this study we have tested the concept of neuron protection by acute bioenergetic intervention or by pharmacological preconditioning with natural antioxidants. Adenosine triphosphate (ATP), pentobarbital, and suramin were encapsulated in pH-sensitive liposomes and used as bioenergy stabilizer. We induced ATP depletion model by incubating cells with media added with ATP-depleting agents for 2 hours. Treatment with bioenergy stabilizer started 10-min post inducing of ATP-depletion. The acute treatment with bioenergy stabilizer significantly increased cell viability in neuro-2a cells. In searching for a pharmacological preconditioning candidate for reducing ischemic injury, we tested cocoa-derived flavanols using bilateral common carotid artery occlusion (BCCAO). We pretreated mice with cocoa-derived flavanols (75 mg/kg) or water orally for 7 days and subjected mice for 12 minutes BCCAO. At 7 days post-ischemia, the number of surviving hippocampal CA1 neurons was significantly higher in the treated mice than in the water-treated controls. The protection from cocoa-derived flavanols was found associated with increased total antioxidant capacity in the brain. Our results indicate that for reducing acute ischemic injury bioenergetic intervention using advanced drug delivery tools is conceptually feasible, and for reducing reperfusion related secondary injury pharmacological preconditioning may provide significant protection.
RESUMO
It is postulated that inhibition of cytosolic phospholipase A(2) alpha (cPLA(2)α) can reduce severity of stroke injury. This is supported by the finding that cPLA(2)α-deficient mice are partially protected from transient, focal cerebral ischemia. The object of this study was to determine the effect of cPLA(2)α inhibition with arachidonyl trifluoromethyl ketone (ATK) on stroke injury in mice. Male C57BL/6 mice were subjected to 1h of focal cerebral ischemia followed by 24 or 72 h of reperfusion. Mice were treated with ATK or vehicle by intermittent intraperitoneal injection or continuous infusion via an implanted infusion pump. ATK injections 1h before and then 1 and 6h after the start of reperfusion significantly reduced infarction volumes in striatum and hemisphere after 24h of reperfusion. ATK did not reduce injury if it was not administered before onset of ischemia or was not administered after 6h of reperfusion. Intermittent doses of ATK failed to reduce infarct volume after 72 h of reperfusion. Continuous infusion with ATK throughout 72h of reperfusion significantly reduced cortical and whole hemispheric infarct volume compared to vehicle treatment. Following ischemia and reperfusion, ATK treatment significantly reduced brain PLA(2) activity. These results are the first to demonstrate a therapeutic effect of cPLA(2)α inhibition on ischemia and reperfusion injury and define a therapeutic time window. cPLA(2)α activity augments injury in the acute and delayed phases of cerebral ischemia and reperfusion injury. We conclude that cPLA(2)α inhibition may be clinically useful if started before initiation of cerebral ischemia.
Assuntos
Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/deficiência , Infarto da Artéria Cerebral Média/complicações , Análise de Variância , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Ácidos Araquidônicos/uso terapêutico , Temperatura Corporal/efeitos dos fármacos , Infarto Encefálico/etiologia , Infarto Encefálico/prevenção & controle , Lesões Encefálicas/tratamento farmacológico , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Lateralidade Funcional/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrobenzenos/uso terapêutico , Reperfusão , Traumatismo por Reperfusão/prevenção & controle , Sulfonamidas/uso terapêutico , Fatores de TempoRESUMO
Stroke and Alzheimer's disease (AD) are major age-related neurodegenerative diseases that may worsen the prognosis of each other. Our study was designed to delineate the prostaglandin E(2) EP1 receptor role in AD and in the setting of cerebral ischemia. Genetic deletion of the prostaglandin EP1 receptor significantly attenuated the more severe neuronal damage (38.5 ± 10.6%) and memory loss induced by ischemic insult observed in AD transgenic mice (percentage of viable hippocampal CA1 neurons: 11.2 ± 2.9%) when compared with wild type mice (45.1 ± 9.1%). In addition, we found that the amyloid plaques were reduced in EP1 deleted AD mice. ß-amyloid-induced toxicity (18.0 ± 7.1%) and Ca(2+) response (91.8 ± 12.9%) were also reduced in EP1(-/-) neurons compared with control neurons in in vitro. Hence, EP1 might mediate most of the toxicity associated with cyclooxygenase-2 and contribute substantially to the cell death pathways in AD and stroke. Exploring potential therapeutic agent targeting EP1 receptor could potentially benefit treatments for stroke and AD patients.
Assuntos
Doença de Alzheimer/complicações , Isquemia Encefálica/complicações , Síndromes Neurotóxicas/complicações , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Encéfalo/citologia , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Glucose/deficiência , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Hidrazinas/farmacologia , Hipóxia/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Síndromes Neurotóxicas/etiologia , Oxazepinas/farmacologia , Fragmentos de Peptídeos/toxicidade , Presenilina-1/genética , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP1/deficiênciaRESUMO
Interest in histone deacetylase (HDAC)-based therapeutics as a potential treatment for stroke has grown dramatically. The neuroprotection of HDAC inhibition may involve multiple mechanisms, including modulation of transcription factor acetylation independent of histones. The transcription factor Nrf2 has been shown to be protective in stroke as a key regulator of antioxidant-responsive genes. Here, we hypothesized that HDAC inhibition might provide neuroprotection against mouse cerebral ischemia by activating the Nrf2 pathway. We determined that the classic HDAC inhibitor trichostatin A increased neuronal cell viability after oxygen-glucose deprivation (from an OD value of 0.10±0.01 to 0.25±0.08) and reduced infarct volume in wild-type mice with stroke (from 49.1±3.8 to 21.3±4.6%). In vitro studies showed that HDAC inhibition reduced Nrf2 suppressor Keap1 expression, induced Keap1/Nrf2 dissociation, Nrf2 nuclear translocation, and Nrf2 binding to antioxidant response elements in heme oxygenase 1 (HO1), and caused HO1 transcription. Furthermore, we demonstrated that HDAC inhibition upregulated proteins downstream of Nrf2, including HO1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit in neuron cultures and brain tissue. Finally, unlike wild-type mice, Nrf2-deficient mice were not protected by pharmacologic inhibition of HDAC after cerebral ischemia. Our studies suggest that activation of Nrf2 might be an important mechanism by which HDAC inhibition provides neuroprotection.