RESUMO
BACKGROUND: High mobility group box 1 protein (HMGB1) is an alarmin following its release by immune cells upon cellular activation or stress. High levels of extracellular HMGB1 play a critical role in impairing the clearance of invading pulmonary pathogens and dying neutrophils in the injured lungs of cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS). A heparin derivative, 2-O, 3-O desulfated heparin (ODSH), has been shown to inhibit HMGB1 release from a macrophage cell line and is efficacious in increasing bacterial clearance in a mouse model of pneumonia. Thus, we hypothesized that ODSH can attenuate the bacterial burden and inflammatory lung injury in CF and we conducted experiments to determine the underlying mechanisms. METHODS: We determined the effects of ODSH on lung injury produced by Pseudomonas aeruginosa (PA) infection in CF mice with the transmembrane conductance regulator gene knockout (CFTR-/-). Mice were given ODSH or normal saline intraperitoneally, followed by the determination of the bacterial load and lung injury in the airways and lung tissues. ODSH binding to HMGB1 was determined using surface plasmon resonance and in silico docking analysis of the interaction of the pentasaccharide form of ODSH with HMGB1. RESULTS: CF mice given 25 mg/kg i.p. of ODSH had significantly lower PA-induced lung injury compared to mice given vehicle alone. The CF mice infected with PA had decreased levels of nitric oxide (NO), increased levels of airway HMGB1 and HMGB1-impaired macrophage phagocytic function. ODSH partially attenuated the PA-induced alteration in the levels of NO and airway HMGB1 in CF mice. In addition, ODSH reversed HMGB1-impaired macrophage phagocytic function. These effects of ODSH subsequently decreased the bacterial burden in the CF lungs. In a surface plasmon resonance assay, ODSH interacted with HMGB1 with high affinity (KD = 3.89 × 10-8 M) and induced conformational changes that may decrease HMGB1's binding to its membrane receptors, thus attenuating HMGB1-induced macrophage dysfunction. CONCLUSIONS: The results suggest that ODSH can significantly decrease bacterial infection-induced lung injury in CF mice by decreasing both HMGB1-mediated impairment of macrophage function and the interaction of HMGB1 with membrane receptors. Thus, ODSH could represent a novel approach for treating CF and ARDS patients that have HMGB1-mediated lung injury.
Assuntos
Fibrose Cística/complicações , Fibrose Cística/metabolismo , Proteína HMGB1/genética , Heparina/análogos & derivados , Macrófagos/imunologia , Macrófagos/metabolismo , Pneumonia Bacteriana/etiologia , Pneumonia Bacteriana/metabolismo , Animais , Carga Bacteriana , Biomarcadores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Heparina/química , Heparina/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Óxido Nítrico/metabolismo , Fagocitose/imunologia , Pneumonia Bacteriana/patologia , Ligação Proteica , Células RAW 264.7 , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Disulfiram and metals inactivate key oncoproteins resulting in anti-neoplastic activity. The goal of this study was to determine the maximum tolerated dose of copper when administered with disulfiram in patients with advanced solid tumors and liver involvement. METHODS: Disulfiram 250 mg was administered daily in 28-day cycles. Four doses of copper gluconate were tested (2, 4, 6, and 8 mg of elemental copper) in a standard 3 + 3 dose escalation design. Patients were evaluated for dose limiting toxicities and response. Protein S-glutathionylation was evaluated as a pharmacodynamic marker. RESULTS: Twenty-one patients were enrolled and 16 patients were evaluable for dose limiting toxicities. Among the 21 patients, there was a median of 4 lines of prior chemotherapy. Five Grade 3 toxicities were observed (anorexia, elevated aspartate aminotransferase or AST, elevated alkaline phosphatase, fever, and fatigue). Response data was available for 15 patients. Four patients had stable disease with the longest duration of disease control being 116 days. The median duration of treatment for evaluable patients was 55 days (range 28-124). Reasons for discontinuation included functional decline, disease progression, and disease-associated death. Increased S-glutathionylation of serum proteins was observed with treatment. CONCLUSION: Disulfiram 250 mg daily with copper gluconate (8 mg of elemental copper) was well-tolerated in patients with solid tumors involving the liver and was not associated with dose limiting toxicities. While temporary disease stabilization was noted in some patients, no objective responses were observed. Treatment was associated with an increase in S-glutathionylation suggesting that this combination could exert a suppressive effect on cellular growth and protein function. TRIAL REGISTRATION: NCT00742911 , first posted 28/08/2008.
Assuntos
Dissulfiram/administração & dosagem , Gluconatos/administração & dosagem , Glutationa/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Dissulfiram/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Gluconatos/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismoRESUMO
The coronavirus SARS-CoV-2 of 2019 (COVID-19) causes a pandemic that has been diagnosed in more than 70 million people worldwide. Mild-to-moderate COVID-19 symptoms include coughing, fever, myalgia, shortness of breath, and acute inflammatory lung injury (ALI). In contrast, acute respiratory distress syndrome (ARDS) and respiratory failure occur in patients diagnosed with severe COVID-19. ARDS is mediated, at least in part, by a dysregulated inflammatory response due to excessive levels of circulating cytokines, a condition known as the "cytokine-storm syndrome." Currently, there are FDA-approved therapies that attenuate the dysregulated inflammation that occurs in COVID-19 patients, such as dexamethasone or other corticosteroids and IL-6 inhibitors, including sarilumab, tocilizumab, and siltuximab. However, the efficacy of these treatments have been shown to be inconsistent. Compounds that activate the vagus nerve-mediated cholinergic anti-inflammatory reflex, such as the α7 nicotinic acetylcholine receptor agonist, GTS-21, attenuate ARDS/inflammatory lung injury by decreasing the extracellular levels of high mobility group box-1 (HMGB1) in the airways and the circulation. It is possible that HMGB1 may be an important mediator of the "cytokine-storm syndrome." Notably, high plasma levels of HMGB1 have been reported in patients diagnosed with severe COVID-19, and there is a significant negative correlation between HMGB1 plasma levels and clinical outcomes. Nicotine can activate the cholinergic anti-inflammatory reflex, which attenuates the up-regulation and the excessive release of pro-inflammatory cytokines/chemokines. Therefore, we hypothesize that low molecular weight compounds that activate the cholinergic anti-inflammatory reflex, such as nicotine or GTS-21, may represent a potential therapeutic approach to attenuate the dysregulated inflammatory responses in patients with severe COVID-19.
Assuntos
Compostos de Benzilideno/farmacologia , Tratamento Farmacológico da COVID-19 , Colinérgicos/farmacologia , Inflamação/tratamento farmacológico , Nicotina/metabolismo , Piridinas/farmacologia , SARS-CoV-2/fisiologia , Tabagismo/tratamento farmacológico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fumar Cigarros/efeitos adversos , Dexametasona/uso terapêutico , Proteína HMGB1/sangue , Humanos , Pandemias , Receptor Nicotínico de Acetilcolina alfa7/agonistasRESUMO
PURPOSE: Oral mucositis (OM) is a common, painful side effect of radiation therapy used for the treatment of head and neck cancer (HNC). Activation of the innate immune system upon irradiation has been identified as a key precipitating event of OM. To better understand OM's pathogenesis, we studied pattern recognition receptors (PRRs) and their downstream pro-inflammatory cytokines in a mouse model of radiation-induced OM. We also tested therapeutic efficacy of GM-1111 that targets innate immune system to reduce radiation-induced OM. METHODS AND MATERIALS: The pathogenesis of OM was studied in a single X-ray induced mouse model. The severity of OM was measured by visual and microscopical examinations. The irradiation-induced changes of PRRs and their downstream effector cytokine gene expression levels were determined. The efficacy of GM-1111 to reduce OM was tested in single and fractionated irradiation mouse models. The impact of the drug on tumor response to radiation therapy was also tested in a mouse model of human HNC. RESULTS: Radiation-induced tissue ulcerations were radiation-dosage and -time dependent. The lesions showed selective increases in PRR and pro-inflammatory cytokine gene expression levels. Once daily administration of GM-1111 (≥30 mg/kg, s.c.) significantly reduced the severity and the incidence of OM. The drug had little effect on PRRs but significantly inhibited downstream pro-inflammatory cytokine genes. GM-1111 did not interfere radiation therapy to induce HNC SCC-25 tumor regression. Instead, we observed significant drug-induced tumor regression. CONCLUSIONS: Radiation induces tissue damages. The increased expression levels of PRRs and their downstream pro-inflammatory cytokine genes in the damaged tissues suggest their important contribution to the pathogenesis of OM. Drug GM-1111 that targets these innate immune molecules may be a potential drug candidate as an intervention for OM.
Assuntos
Indanos/farmacologia , Lesões por Radiação/tratamento farmacológico , Lesões por Radiação/patologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estomatite/tratamento farmacológico , Estomatite/patologia , Tiazóis/farmacologia , Animais , Inflamação/patologia , Masculino , Camundongos , Terapia de Alvo Molecular , Transdução de Sinais/efeitos da radiaçãoAssuntos
COVID-19 , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Heparina/administração & dosagem , Programas de Rastreamento/métodos , Trombofilia , Tromboembolia Venosa , Anticoagulantes/administração & dosagem , COVID-19/sangue , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/fisiopatologia , Quimioprevenção/métodos , Cuidados Críticos/métodos , Feminino , Necessidades e Demandas de Serviços de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Processos e Resultados em Cuidados de Saúde , Prevalência , Medição de Risco/métodos , Fatores de Risco , Sensibilidade e Especificidade , Trombofilia/diagnóstico , Trombofilia/virologia , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/epidemiologia , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/prevenção & controleRESUMO
Cystic fibrosis (CF) lung disease is marked by high concentrations of neutrophil elastase (NE) and DNA polymers; both factors contribute to airway disease. Although inhaled recombinant human dornase alfa reduces the frequency of CF pulmonary exacerbations, it also increases free NE activity in the sputum. There are no approved anti-NE therapies for patients with CF. We investigated whether synthetic, low-molecular weight polysulfated hyaluronan GlycoMira-1111 (GM-1111) would be effective as an anti-NE drug using ex vivo CF sputum. Anti-NE activity of GM-1111 was tested in CF sputum in the presence or absence of dornase alfa and/or hypertonic saline using a spectrophotometric assay specific for human NE and was compared with unfractionated heparin. We tested whether GM-1111 disaggregated DNA from CF sputum (using gel electrophoresis analysis) or modified CF sputum viscoelastic properties (using a dynamic rheometer). GM-1111 and unfractionated heparin had near equivalent anti-NE activity in CF sputum in the presence of dornase alfa. Both GM-1111 and unfractionated heparin retained anti-NE activity in hypertonic saline but with decreased activity. GM-1111 increased the release of soluble DNA in CF sputum, resulting in improved depolymerization efficacy of dornase alfa. GM-1111 decreased CF sputum elasticity. GM-1111 inhibited NE activity, enhanced DNA depolymerization by deoxyribonuclease, and decreased viscoelastic properties of CF sputum, similar to effects reported previously for unfractionated heparin. Unlike heparins, GM-1111 is synthetic, with minimal anticoagulant activity, and is not derived from animal products. These key attributes provide advantages over unfractionated heparin as a potential therapeutic for CF.
Assuntos
Fibrose Cística/tratamento farmacológico , Ácido Hialurônico/uso terapêutico , Elastase de Leucócito/metabolismo , Escarro/efeitos dos fármacos , Escarro/metabolismo , Adulto , Anti-Inflamatórios/uso terapêutico , Fibrose Cística/metabolismo , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Feminino , Heparina/uso terapêutico , Humanos , Masculino , Proteínas Recombinantes/metabolismo , ReologiaRESUMO
Chronic rhinosinusitis (CRS) is characterized by sustained mucosal inflammation, impaired mucociliary clearance, loss of cilia and epithelial barrier breakdown, and tissue remodeling. Certain glycosaminoglycans inhibit various inflammatory mediators, suppress bacterial growth, and provide important functions in mucosal tissue repair and mucociliary clearance. Herein, we evaluated the effects of a synthetic glycosaminoglycan, GM-1111, on the clinical signs and inflammatory tissue changes associated with CRS in mice. CRS was generated by repeated intranasal applications of Aspergillus fumigatus (A. fumigatus) extracts over 4 weeks. Mice were then intranasally administered GM-1111 (600 µg per dose, 5 times a week) or vehicle (phosphate buffered saline, PBS) for an additional 4 weeks while still being given A. fumigatus extracts to maintain a chronic inflammatory environment with acute exacerbations. Clinical signs indicative of sinonasal inflammation were recorded throughout the study. After 9 weeks, whole blood and sinonasal tissues were harvested for hematological, histological, and biochemical examination. The clinical signs, white blood cell counts, tissue markers of sinonasal inflammation, and histological changes caused by A. fumigatus extract administration were compared to the healthy (PBS vehicle) and GM-1111-treated groups (n = 12 per treatment group). Compared to vehicle-treated animals, animals treated with GM-1111 demonstrated significant reductions in clinical signs (p<0.05), degenerative tissue changes, goblet cell hyperplasia, inflammatory cell infiltration (p<0.01), innate immunity- (tlr2, tlr4, myd88, il1b, tnfa, il6, and il12) and adaptive immunity-associated (ccl11, ccl24, ccl5, il4, il5, and il13) cytokine gene expression (p<0.05 to p<0.0001) in sinonasal tissues, and serum IgE levels (p<0.01). Our data suggest that GM-1111 significantly reduces local and systemic effects of CRS-associated sinonasal inflammation.
Assuntos
Anti-Inflamatórios/uso terapêutico , Glicosaminoglicanos/uso terapêutico , Inflamação/tratamento farmacológico , Rinite/tratamento farmacológico , Sinusite/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Doença Crônica , Citocinas/análise , Citocinas/imunologia , Modelos Animais de Doenças , Glicosaminoglicanos/química , Imunoglobulina E/análise , Imunoglobulina E/imunologia , Inflamação/complicações , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos Endogâmicos BALB C , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Mucosa Nasal/patologia , Rinite/complicações , Rinite/imunologia , Rinite/patologia , Sinusite/complicações , Sinusite/imunologia , Sinusite/patologiaRESUMO
Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4's effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4-/- murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT.
Assuntos
Heparina/análogos & derivados , Trombocitopenia/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Heparina/farmacologia , Heparina/uso terapêutico , Humanos , Megacariócitos/citologia , Camundongos , Contagem de Plaquetas , Fator Plaquetário 4/sangue , Fator Plaquetário 4/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/etiologia , TrombopoeseRESUMO
Relapses in acute myelogenous leukemia (AML) are a result of quiescent leukemic stem cells (LSCs) in marrow stromal niches, where they resist chemotherapy. LSCs employ CXCL12/CXCR4 to home toward protective marrow niches. Heparin disrupts CXCL12-mediated sequestration of cells in the marrow. CX-01 is a low-anticoagulant heparin derivative. In this pilot study, we combined CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7 + 3) with CX-01. Twelve patients were enrolled (median age, 56 years; 3 women). Three, 5, and 4 patients had good-, intermediate-, and poor-risk disease, respectively. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had morphologic complete remission after 1 induction (CR1). Eight patients were alive at a median follow-up of 24 months (4 patients in CR1). Three patients received an allogeneic stem cell transplant in CR1. Median disease-free survival was 14.8 months. Median overall survival was not attained at the maximum follow-up time of 29.4 months. No CX-01-associated serious adverse events occurred. Median day to an untransfused platelet count of at least 20 × 109/L was 21. CX-01 is well tolerated when combined with intensive therapy for AML and appears associated with enhanced count recovery and treatment efficacy.
Assuntos
Antineoplásicos/uso terapêutico , Quimioterapia Combinada/métodos , Heparina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Adulto , Idoso , Anticoagulantes/uso terapêutico , Feminino , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. METHODS: We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. RESULTS: (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1-10 ng/mL vs. > 100 µg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. CONCLUSIONS: We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.
Assuntos
Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Biofilmes/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Glicosaminoglicanos/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Aggregatibacter actinomycetemcomitans/metabolismo , Animais , Anti-Inflamatórios não Esteroides/síntese química , Sítios de Ligação , Biofilmes/crescimento & desenvolvimento , Conservadores da Densidade Óssea/síntese química , Linhagem Celular , Expressão Gênica , Glicosaminoglicanos/síntese química , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Periodontite/prevenção & controle , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/metabolismo , Ligação Proteica , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Stress (Takotsubo) cardiomyopathy is a form of reversible left ventricular dysfunction with a heightened risk of ventricular arrhythmia thought to be caused by high circulating catecholamines. We report a case of stress cardiomyopathy that developed during severe alcohol withdrawal successfully treated with dexmedetomidine. The case involves a 53-year-old man with a significant history of alcohol abuse who presented to a teaching hospital with new-onset seizures. His symptoms of acute alcohol withdrawal were initially treated with benzodiazepines, but the patient later developed hypotension, and stress cardiomyopathy was suspected based on ECG and echocardiographic findings. Adjunctive treatment with the alpha-2-adrenergic agonist, dexmedetomidine, was initiated to curtail excessive sympathetic outflow of the withdrawal syndrome, thereby targeting the presumed pathophysiology of the cardiomyopathy. Significant clinical improvement was observed within one day of initiation of dexmedetomidine. These findings are consistent with other reports suggesting that sympathetic dysregulation during alcohol withdrawal produces ideal pathobiology for stress cardiomyopathy and leads to ventricular arrhythmogenicity. Stress cardiomyopathy should be recognized as a complication of alcohol withdrawal that significantly increases cardiac-related mortality. By helping to correct autonomic dysregulation of the withdrawal syndrome, dexmedetomidine may be useful in the treatment of stress-induced cardiomyopathy.
RESUMO
Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhance lung inflammation, activate endothelial cells and disrupt lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (ECs). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin was also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH can interfere with thrombin-activated signaling.
Assuntos
Anticoagulantes/farmacologia , Células Endoteliais/efeitos dos fármacos , Heparina/análogos & derivados , Permeabilidade/efeitos dos fármacos , Receptores de Trombina/metabolismo , Trombina/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Heparina/farmacologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Neutrophil elastase (NE) is a major inflammatory mediator in cystic fibrosis (CF) that is a robust predictor of lung disease progression. NE directly causes airway injury via protease activity, and propagates persistent neutrophilic inflammation by up-regulation of neutrophil chemokine expression. Despite its key role in the pathogenesis of CF lung disease, there are currently no effective antiprotease therapies available to patients with CF. Although heparin is an effective antiprotease and anti-inflammatory agent, its anticoagulant activity prohibits its use in CF, due to risk of pulmonary hemorrhage. In this report, we demonstrate the efficacy of a 2-O, 3-O-desulfated heparin (ODSH), a modified heparin with minimal anticoagulant activity, to inhibit NE activity and to block NE-induced airway inflammation. Using an established murine model of intratracheal NE-induced airway inflammation, we tested the efficacy of intratracheal ODSH to block NE-generated neutrophil chemoattractants and NE-triggered airway neutrophilic inflammation. ODSH inhibited NE-induced keratinocyte-derived chemoattractant and high-mobility group box 1 release in bronchoalveolar lavage. ODSH also blocked NE-stimulated high-mobility group box 1 release from murine macrophages in vitro, and inhibited NE activity in functional assays consistent with prior reports of antiprotease activity. In summary, this report suggests that ODSH is a promising antiprotease and anti-inflammatory agent that may be useful as an airway therapy in CF.
Assuntos
Anti-Inflamatórios/farmacologia , Proteína HMGB1/metabolismo , Heparina/análogos & derivados , Elastase de Leucócito/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Inibidores de Proteases/farmacologia , Animais , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Heparina/farmacologia , Interleucina-13 , Elastase de Leucócito/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/fisiopatologiaRESUMO
Nosocomial pneumonia (NP, or hospital-acquired pneumonia) is associated with infections originating from hospital-borne pathogens. Persistent microbial presence and acute lung injury are common features of these infections, contributing to the high mortality rates and excessive financial burden for these patients. Pseudomonas aeruginosa (PA), a gram-negative opportunistic pathogen, is one of the prominent pathogens associated with NP. PA pneumonia is characterized by excessive secretion of inflammatory cytokines, neutrophil infiltration, and subsequent lung damage. The persistent presence of PA along with overwhelming inflammatory response is suggestive of impairment in innate immunity. High mobility group box 1 (HMGB1), a recently discovered potent pro-inflammatory cytokine, plays an important role in PA lung infections by compromising innate immunity via impairing phagocyte function through toll-like receptors (TLR) TLR2 and TLR4. ODSH (2-O, 3-O-desulfated heparin), a heparin derivative with significant anti-inflammatory properties but minimal anti-coagulatory effects, has been shown to reduce neutrophilic lung injury in the absence of active microbial infections. This study examined the effects of ODSH on PA pneumonia. This study demonstrates that ODSH not only reduced PA-induced lung injury, but also significantly increased bacterial clearance. The ameliorated lung injury, together with the increased bacterial clearance, resulted in marked improvement in the survival of these animals. The resulting attenuation in lung injury and improvement in bacterial clearance were associated with decreased levels of airway HMGB1. Furthermore, binding of HMGB1 to its receptors TLR2 and TLR4 was blunted in the presence of ODSH. These data suggest that ODSH provides a potential novel approach in the adjunctive treatment of PA pneumonia.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Infecção Hospitalar/tratamento farmacológico , Heparina/análogos & derivados , Pulmão/efeitos dos fármacos , Pneumonia Pneumocócica/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Animais , Carga Bacteriana/efeitos dos fármacos , Infecção Hospitalar/imunologia , Modelos Animais de Doenças , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Heparina/administração & dosagem , Humanos , Imunidade Inata/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/imunologia , Infecções por Pseudomonas/imunologia , Regulação para CimaRESUMO
PURPOSE: Studies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation. MATERIALS AND METHODS: C57BL/6 mice were catheterized/instilled with LL-37 (320 µM, 150 µl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy, experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate. RESULTS: Profound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature. CONCLUSIONS: We developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation.
Assuntos
Peptídeos Catiônicos Antimicrobianos/toxicidade , Cistite Intersticial/tratamento farmacológico , Glicosaminoglicanos/uso terapêutico , Bexiga Urinária/patologia , Administração Intravesical , Animais , Peptídeos Catiônicos Antimicrobianos/química , Cromatografia Líquida de Alta Pressão , Cistite Intersticial/induzido quimicamente , Cistite Intersticial/patologia , Modelos Animais de Doenças , Feminino , Glicosaminoglicanos/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Bexiga Urinária/efeitos dos fármacos , CatelicidinasRESUMO
Heparin which is desulfated at the 2-O and 3-O positions (ODSH) has reduced anti-coagulant properties, and reduced interaction with heparin antibodies. Because of the reduced anti-coagulant effect, ODSH can be safely administered to animals and humans intravenously at doses up to 20 mg/kg, resulting in a serum concentration of up to 250µg/ml. Administration of ODSH causes a 35% reduction in infarct size in dogs and pigs subjected to coronary artery occlusion and reperfusion when given 5 min before reperfusion. ODSH has anti-inflamatory effects, manifest as a decrease in neutrophil infiltration into ischemic tissue at high doses, but this effect does not entirely account for the reduction in infarct size. ODSH decreases Na(+) and Ca(2+) loading in isolated cardiac myocytes subjected to simulated ischemia. This effect appears due to an ODSH-induced reduction in an enhanced Na(+) influx via the Na channel in the membrane of cardiac myocyes caused by oxygen radicals generated during ischemia and reperfusion. Reduction in Na(+) influx decreases Ca(2+) loading by reducing Ca2(+) influx via Na/Ca exchange, thus reducing Ca(2+) - dependent reperfusion injury. ODSH does not appear to interact with antibodies to the heparin/platelet factor 4 complex, and does not cause heparin-induced thrombocytopenia. Because of these therapeutic and safety considerations, ODSH would appear to be a promising heparin derivative for prevention of reperfusion injury in humans undergoing thrombolytic or catheter-based reperfusion for acute myocardial infarction. The review article discussed the use of heparin and the discussion of some of the important patents, including: US6489311; US7478358; PCTUS2008070836 and PCTUS2009037836.
Assuntos
Anticoagulantes/farmacologia , Heparina/análogos & derivados , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacologia , Anticoagulantes/efeitos adversos , Modelos Animais de Doenças , Cães , Heparina/efeitos adversos , Heparina/farmacologia , Humanos , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Patentes como Assunto , SuínosRESUMO
BACKGROUND: Rosacea is a common disfiguring skin disease of primarily Caucasians characterized by central erythema of the face, with telangiectatic blood vessels, papules and pustules, and can produce skin thickening, especially on the nose of men, creating rhinophyma. Rosacea can also produce dry, itchy eyes with irritation of the lids, keratitis and corneal scarring. The cause of rosacea has been proposed as over-production of the cationic cathelicidin peptide LL-37. METHODOLOGY/PRINCIPAL FINDINGS: We tested a new class of non-anticoagulant sulfated anionic polysaccharides, semi-synthetic glycosaminoglycan ethers (SAGEs) on key elements of the pathogenic pathway leading to rosacea. SAGEs were anti-inflammatory at ng/ml, including inhibition of polymorphonuclear leukocyte (PMN) proteases, P-selectin, and interaction of the receptor for advanced glycation end-products (RAGE) with four representative ligands. SAGEs bound LL-37 and inhibited interleukin-8 production induced by LL-37 in cultured human keratinocytes. When mixed with LL-37 before injection, SAGEs prevented the erythema and PMN infiltration produced by direct intradermal injection of LL-37 into mouse skin. Topical application of a 1% (w/w) SAGE emollient to overlying injected skin also reduced erythema and PMN infiltration from intradermal LL-37. CONCLUSIONS: Anionic polysaccharides, exemplified by SAGEs, offer potential as novel mechanism-based therapies for rosacea and by extension other LL-37-mediated and RAGE-ligand driven skin diseases.
Assuntos
Catelicidinas/metabolismo , Glicosaminoglicanos/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Rosácea/tratamento farmacológico , Rosácea/metabolismo , Sulfatos/química , Administração Tópica , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Óleo de Cróton/efeitos adversos , Modelos Animais de Doenças , Glicosaminoglicanos/administração & dosagem , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/uso terapêutico , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Ligantes , Antígeno de Macrófago 1/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Selectina-P/metabolismo , Peptídeo Hidrolases/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Rosácea/induzido quimicamenteRESUMO
While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.