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1.
Respiration ; 98(1): 60-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30799409

RESUMO

BACKGROUND: Granulation tissue is a common complication of airway stenting, but no published methods can quantify the volume and type of tissue that develops. OBJECTIVE: To use design-based stereology to quantify changes in tissue volume and type associated with airway stenting. METHODS: We compared drug-eluting stents (DES) filled with gendine to standard silicone stents in pigs in an assessor-blinded randomized trial. Tracheal stents were placed via rigid bronchoscopy. After 1 month, animals were euthanized and necropsies were performed. Antimicrobial effects of the DES were assessed in trachea tissue samples, on the DES surface, and with residual gel from the DES reservoir. Tracheal thickness was measured using orthogonal intercepts. Design-based stereology was used to quantify the volume density of tissues using a point-counting method. The volume of each tissue was normalized to cartilage volume, which is unaffected by stenting. RESULTS: Pigs were randomized to DES (n = 36) or control stents (n = 9). The drug was successfully eluted from the DES, and the stent surface showed antibacterial activity. DES and controls did not differ in tissue microbiology, tracheal thickness, or granulation tissue volume. Compared to nonstented controls, stented airways demonstrated a 110% increase in soft-tissue volume (p = 0.005). Submucosal connective tissue (118%; p < 0.0001), epithelium (70%; p < 0.0001), submucosal glands (47%; p = 0.001), and smooth muscle (41%; p < 0.0001) increased in volume. CONCLUSION: Stenting doubles the volume of soft tissue in the trachea. Design-based stereology can quantify the tissue changes associated with airway stenting.

2.
JCI Insight ; 4(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30721150

RESUMO

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell-deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.

3.
J Biol Chem ; 294(13): 4784-4792, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696774

RESUMO

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

4.
J Biol Chem ; 294(9): 3012-3023, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30563839

RESUMO

Mast cells (MCs) participate in allergy, inflammation, and defense against pathogens. They release multiple immune mediators via exocytosis, a process that requires SNARE proteins, including syntaxins (Stxs). The identity of the Stxs involved in MC exocytosis remains controversial. Here, we studied the roles of Stx3 and -4 in fully developed MCs from conditional knockout mice by electrophysiology and EM, and found that Stx3, and not Stx4, is crucial for MC exocytosis. The main defect seen in Stx3-deficient MCs was their inability to engage multigranular compound exocytosis, while leaving most single-vesicle fusion events intact. We used this defect to show that this form of exocytosis is not only required to accelerate MC degranulation but also essential to achieve full degranulation. The exocytic defect was severe but not absolute, indicating that an Stx other than Stx3 and -4 is also required for exocytosis in MCs. The removal of Stx3 affected only regulated exocytosis, leaving other MC effector responses intact, including the secretion of cytokines via constitutive exocytosis. Our in vivo model of passive systemic anaphylaxis showed that the residual exocytic function of Stx3-deficient MCs was sufficient to drive a full anaphylactic response in mice.


Assuntos
Exocitose , Mastócitos/citologia , Proteínas Qa-SNARE/metabolismo , Animais , Contagem de Células , Degranulação Celular , Diferenciação Celular , Técnicas de Inativação de Genes , Cinética , Camundongos , Proteínas Qa-SNARE/deficiência , Proteínas Qa-SNARE/genética
5.
J Bronchology Interv Pulmonol ; 25(4): 322-329, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29771774

RESUMO

BACKGROUND: Population aging and lung cancer screening strategies may lead to an increase in detection of early-stage lung cancer in medical inoperable patients. Recent advances in peripheral bronchoscopy have made it a suitable platform for ablation of small peripheral tumors. METHODS: We investigated the tissue-ablative effect of a diode laser bronchoscopically applied by a laser delivery fiber (LDF) with wide aperture on porcine lung parenchyma. Laser was tested ex vivo and in vivo to identify the most effective power settings and LDF. Chest computed tomography (CT) were obtained immediately after ablation and after 3 days of observation. At day 3, necropsy was performed. RESULTS: On the basis of our ex vivo and in vivo experiments, we selected the round-tip LDF to be activated at 25 W for 20 seconds. Ten ablations were performed in 5 pigs. One ablation resulted in a pneumothorax requiring aspiration. All animals remained stable for 72 hours. CT findings at days 1 and 3 showed an area of cavitation surrounded by consolidation and ground glass. Median size of CT findings (long axis) was 26 mm (range, 24 to 38) at day 1, and 34 mm (range, 30 to 44) at day 3. Necropsy showed an area of central char measuring from 0.8×0.7×0.9 cm to 2.4×3.5×1.2 cm, surrounded by a gray-brown to dark red area. On histology, variable degrees of necrosis were evident around the charred areas. CONCLUSION: Bronchoscopic laser interstitial thermal therapy can achieve relatively large areas of ablation of normal lung parenchyma with a low rate of periprocedural complications.

6.
Haematologica ; 103(7): 1235-1244, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29674495

RESUMO

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4. We assessed platelet granule exocytosis in Munc13-2 and -4 global and conditional knockout (KO) mice, and observed that deletion of Munc13-4 ablates dense granule release and indirectly impairs alpha granule exocytosis. We found no exocytic role for Munc13-2 in platelets, not even in the absence of Munc13-4. In vitro, Munc13-4-deficient platelets exhibited defective aggregation at low doses of collagen. In a flow chamber assay, we observed that Munc13-4 acted as a rate-limiting factor in the formation of thrombi. In vivo, we observed a dose-dependency between Munc13-4 expression in platelets and both venous bleeding time and time to arterial thrombosis. Finally, in a model of allergic airway inflammation, we found that platelet-specific Munc13-4 KO mice had a reduction in airway hyper-responsiveness and eosinophilic inflammation. Taken together, our results indicate that Munc13-4-dependent platelet dense granule release plays essential roles in hemostasis, thrombosis and allergic inflammation.

7.
J Biol Chem ; 293(19): 7148-7159, 2018 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-29599294

RESUMO

Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2-deficient MCs but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, indicating that Munc18-2 is not essential for the migration, retention, and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC-regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.

8.
J Biol Chem ; 293(1): 345-358, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29141910

RESUMO

Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. In vitro assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events (i.e. granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Anafilaxia , Animais , Modelos Animais de Doenças , Exocitose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mastócitos/metabolismo , Mastócitos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Isoformas de Proteínas , Transporte Proteico
9.
Nature ; 505(7483): 412-6, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24317696

RESUMO

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.


Assuntos
Pulmão/imunologia , Mucina-5B/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Animais , Asma/imunologia , Asma/metabolismo , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Cílios/fisiologia , Orelha Média/imunologia , Orelha Média/microbiologia , Feminino , Inflamação/patologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mucina-5AC/deficiência , Mucina-5AC/metabolismo , Mucina-5B/deficiência , Mucina-5B/genética , Fagocitose , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Staphylococcus aureus/imunologia , Análise de Sobrevida
10.
Open Biol ; 3(11): 130163, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24350389

RESUMO

Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a 'critical period' of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.


Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Neurônios Motores/fisiologia , Receptores Nicotínicos/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação , Animais , Cóclea/fisiologia , Camundongos , Camundongos Knockout , Receptores Nicotínicos/genética , Estereocílios , Sinaptotagmina II/genética , Sinaptotagmina II/fisiologia
11.
J Immunol ; 191(3): 1404-12, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23797671

RESUMO

Mouse mast cell protease (mMCP)-6-null C57BL/6 mice lost less aggrecan proteoglycan from the extracellular matrix of their articular cartilage during inflammatory arthritis than wild-type (WT) C57BL/6 mice, suggesting that this mast cell (MC)-specific mouse tryptase plays prominent roles in articular cartilage catabolism. We used ex vivo mouse femoral head explants to determine how mMCP-6 and its human ortholog hTryptase-ß mediate aggrecanolysis. Exposure of the explants to recombinant hTryptase-ß, recombinant mMCP-6, or lysates harvested from WT mouse peritoneal MCs (PMCs) significantly increased the levels of enzymatically active matrix metalloproteinases (MMP) in cartilage and significantly induced aggrecan loss into the conditioned media, relative to replicate explants exposed to medium alone or lysates collected from mMCP-6-null PMCs. Treatment of cartilage explants with tetramer-forming tryptases generated aggrecan fragments that contained C-terminal DIPEN and N-terminal FFGVG neoepitopes, consistent with MMP-dependent aggrecanolysis. In support of these data, hTryptase-ß was unable to induce aggrecan release from the femoral head explants obtained from Chloe mice that resist MMP cleavage at the DIPEN↓FFGVG site in the interglobular domain of aggrecan. In addition, the abilities of mMCP-6-containing lysates from WT PMCs to induce aggrecanolysis were prevented by inhibitors of MMP-3 and MMP-13. Finally, recombinant hTryptase-ß was able to activate latent pro-MMP-3 and pro-MMP-13 in vitro. The accumulated data suggest that human and mouse tetramer-forming tryptases are MMP convertases that mediate cartilage damage and the proteolytic loss of aggrecan proteoglycans in arthritis, in part, by activating the zymogen forms of MMP-3 and MMP-13, which are constitutively present in articular cartilage.


Assuntos
Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Mastócitos/imunologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Animais , Artrite/metabolismo , Células Cultivadas , Precursores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Inflamação , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Triptases/deficiência , Triptases/genética , Triptases/metabolismo
12.
J Allergy Clin Immunol ; 131(3): 752-62, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23380220

RESUMO

BACKGROUND: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. OBJECTIVES: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. METHODS: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. RESULTS: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. CONCLUSION: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.


Assuntos
Modelos Animais de Doenças , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumaça/efeitos adversos , Triptases/imunologia , Remodelação das Vias Aéreas , Animais , Macrófagos/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Testes de Função Respiratória , Tabaco , Triptases/deficiência , Triptases/genética
13.
Circulation ; 127(4): 476-485, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23266857

RESUMO

BACKGROUND: Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. METHODS AND RESULTS: The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide-induced but not thrombin receptor-activating peptide- or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. CONCLUSIONS: These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.


Assuntos
Agregação Plaquetária/fisiologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Vasculite/metabolismo , Animais , Aterosclerose/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Colágeno/metabolismo , Colágeno/farmacologia , Células HEK293 , Humanos , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipase C gama/metabolismo , Fosforilação/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/genética , Quinase Syk , Trombose/metabolismo
14.
Biochem J ; 446(3): 383-94, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22694344

RESUMO

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.


Assuntos
Células Epiteliais/metabolismo , Genes Essenciais , Mastócitos/metabolismo , Proteínas Munc18/genética , Animais , Modelos Animais de Doenças , Elementos E-Box , Feminino , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Munc18/metabolismo , Anafilaxia Cutânea Passiva/genética , Ratos
15.
J Biol Chem ; 287(24): 20047-55, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22511759

RESUMO

RasGRP4 (Ras guanine nucleotide-releasing protein-4) is an intracellular, calcium-regulated guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor expressed in mast cells (MCs) and their progenitors. To study the function of this signaling protein in inflammatory disorders, a homologous recombination approach was used to create a RasGRP4-null C57BL/6 mouse line. The resulting transgenic animals had normal numbers of MCs in their tissues that histochemically and morphologically resembled those in WT C57BL/6 mice. MCs could also be generated from RasGRP4-null mice by culturing their bone marrow cells in IL-3-enriched conditioned medium. Despite these data, the levels of the transcripts that encode the proinflammatory cytokines IL-1ß and TNF-α were reduced in phorbol 12-myristate 13-acetate-treated MCs developed from RasGRP4-null mice. Although inflammation was not diminished in a Dermatophagoides farinae-dependent model of allergic airway disease, dextran sodium sulfate-induced colitis was significantly reduced in RasGRP4-null mice relative to similarly treated WT mice. Furthermore, experimental arthritis could not be induced in RasGRP4-null mice that had received K/BxN mouse serum. The latter findings raise the possibility that the pharmacologic inactivation of this intracellular signaling protein might be an effective treatment for arthritis or inflammatory bowel disease.


Assuntos
Artrite Experimental/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Transdução de Sinais , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Animais , Antígenos de Dermatophagoides/toxicidade , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Experimental/terapia , Asma/induzido quimicamente , Asma/genética , Asma/metabolismo , Asma/patologia , Carcinógenos/farmacologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Citocinas/farmacologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Camundongos , Camundongos Knockout , Acetato de Tetradecanoilforbol/farmacologia , Fatores ras de Troca de Nucleotídeo Guanina/genética
16.
J Biol Chem ; 287(11): 7834-44, 2012 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-22235124

RESUMO

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-ß. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-ß· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.


Assuntos
Coagulação Sanguínea , Fibrina/metabolismo , Fibrinogênio/metabolismo , Heparina/metabolismo , Mastócitos/enzimologia , Proteólise , Vesículas Secretórias/enzimologia , Trombina/metabolismo , Triptases/metabolismo , Anafilaxia/induzido quimicamente , Anafilaxia/enzimologia , Anafilaxia/genética , Anafilaxia/patologia , Animais , Edema/enzimologia , Edema/genética , Edema/patologia , Fibrina/genética , Fibrinogênio/genética , Heparina/genética , Humanos , Imunoglobulina E/metabolismo , Mastócitos/patologia , Camundongos , Camundongos Knockout , Vesículas Secretórias/genética , Pele/enzimologia , Pele/patologia , Trombina/genética , Triptases/genética
17.
Circ Res ; 108(11): 1316-27, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21493897

RESUMO

RATIONALE: Mast cells (MCs) contribute to the formation of abdominal aortic aneurysms (AAAs) by producing biologically active mediators. Tryptase is the most abundant MC granule protein and participates in MC activation, protease maturation, leukocyte recruitment, and angiogenesis-all processes critical to AAA pathogenesis. OBJECTIVE: To test the hypothesis that tryptase participates directly in AAA formation. METHODS AND RESULTS: Immunohistochemistry demonstrated enhanced tryptase staining in media and adventitia of human and mouse AAA lesions. Serum tryptase levels correlated significantly with the annual expansion rate of AAA before (r = 0.30, P = 0.003) and after (r = 0.29, P = 0.005) adjustment for common AAA risk factors in a patient follow-up study, and associated with risks for later surgical repair or overall mortality before (P = 0.009, P = 0.065) and after (P = 0.004, P = 0.001) the adjustment. Using MC protease-6-deficient mice (Mcpt6(-/-)) and aortic elastase perfusion-induced experimental AAAs, we proved a direct role of this tryptase in AAA pathogenesis. Whereas all wild-type (WT) mice developed AAA at 14 or 56 days postperfusion, Mcpt6(-/-) mice were fully protected. AAA lesions from Mcpt6(-/-) mice had fewer inflammatory and apoptotic cells, and lower chemokine levels, than did those from WT mice. MC from WT mice restored reduced AAA lesions and lesion inflammatory cell content in MC-deficient Kit(W-sh/W-sh) mice, but those prepared from Mcpt6(-/-) mice did not. Mechanistic studies demonstrated that tryptase deficiency affected endothelial cell (EC) chemokine and cytokine expression, monocyte transmigration, smooth-muscle cell apoptosis, and MC and AAA lesion cysteinyl cathepsin expression and activities. CONCLUSIONS: This study establishes the direct participation of MC tryptase in the pathogenesis of experimental AAAs, and suggests that levels of this protease can serve as a novel biomarker for abdominal aortic expansion.


Assuntos
Aneurisma da Aorta Abdominal , Mastócitos/enzimologia , Triptases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Apoptose/fisiologia , Biomarcadores/sangue , Catepsinas/genética , Catepsinas/metabolismo , Modelos Animais de Doenças , Feminino , Granulócitos/imunologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Mutantes , Triptases/genética
18.
Proc Natl Acad Sci U S A ; 108(1): 290-5, 2011 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-21173247

RESUMO

Patients with inflammatory bowel disease (IBD) have increased numbers of human tryptase-ß (hTryptase-ß)-positive mast cells (MCs) in the gastrointestinal tract. The amino acid sequence of mouse mast cell protease (mMCP)-6 is most similar to that of hTryptase-ß. We therefore hypothesized that this mMCP, or the related tryptase mMCP-7, might have a prominent proinflammatory role in experimental colitis. The dextran sodium sulfate (DSS) and trinitrobenzene sulfonic acid (TNBS) colitis models were used to evaluate the differences between C57BL/6 (B6) mouse lines that differ in their expression of mMCP-6 and mMCP-7 with regard to weight loss, colon histopathology, and endoscopy scores. Microarray analyses were performed, and confirmatory real-time PCR, ELISA, and/or immunohistochemical analyses were carried out on a number of differentially expressed cytokines, chemokines, and matrix metalloproteinases (MMPs). The mMCP-6-null mice that had been exposed to DSS had significantly less weight loss as well as significantly lower pathology and endoscopy scores than similarly treated mMCP-6-expressing mice. This difference in colitis severity was confirmed endoscopically in the TNBS-treated mice. Evaluation of the distal colon segments revealed that numerous proinflammatory cytokines, chemokines that preferentially attract neutrophils, and MMPs that participate in the remodeling of the ECM were all markedly increased in the colons of DSS-treated WT mice relative to untreated WT mice and DSS-treated mMCP-6-null mice. Collectively, our data show that mMCP-6 (but not mMCP-7) is an essential MC-restricted mediator in chemically induced colitis and that this tryptase acts upstream of many of the factors implicated in IBD.


Assuntos
Doenças Inflamatórias Intestinais/imunologia , Mastócitos/imunologia , Triptases/imunologia , Animais , Quimiocinas/imunologia , Colo/metabolismo , Colo/patologia , Citocinas/imunologia , Sulfato de Dextrana/toxicidade , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/patologia , Mastócitos/metabolismo , Metaloproteinases da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Reação em Cadeia da Polimerase , Ácido Trinitrobenzenossulfônico/toxicidade , Triptases/metabolismo
19.
J Immunol ; 185(12): 7681-90, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21076070

RESUMO

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.


Assuntos
Queimaduras/imunologia , Quimases/imunologia , Cicatriz/imunologia , Epiderme/imunologia , Mastócitos/imunologia , Modelos Imunológicos , Serina Endopeptidases/imunologia , Animais , Queimaduras/enzimologia , Queimaduras/genética , Queimaduras/patologia , Carboxipeptidases A/genética , Carboxipeptidases A/imunologia , Carboxipeptidases A/metabolismo , Degranulação Celular/genética , Degranulação Celular/imunologia , Quimases/genética , Quimases/metabolismo , Quimases/farmacologia , Cicatriz/enzimologia , Cicatriz/genética , Cicatriz/patologia , Epiderme/enzimologia , Epiderme/patologia , Humanos , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Inflamação , Elastase de Leucócito/genética , Elastase de Leucócito/imunologia , Elastase de Leucócito/metabolismo , Elastase de Leucócito/farmacologia , Mastócitos/enzimologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Miosinas/genética , Miosinas/imunologia , Miosinas/metabolismo , Proteínas Proto-Oncogênicas c-kit , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/farmacologia , Triptases/genética , Triptases/imunologia , Triptases/metabolismo , Triptases/farmacologia
20.
J Med Case Rep ; 3: 9290, 2009 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-19946509

RESUMO

INTRODUCTION: Invasive pulmonary aspergillosis is the most common form of infection by Aspergillus species among immunocompromised patients. Although this infection frequently involves the lung parenchyma, it is unusual to find it limited to the tracheobronchial tree, a condition known as invasive aspergillus tracheobronchitis. CASE PRESENTATION: A 65 year-old Hispanic man from Bolivia with a history of chronic lymphocytic leukemia developed cough and malaise eight months after having an allogenic stem cell transplant. A computed tomography of the chest revealed an area of diffuse soft tissue thickening around the left main stem bronchus, which was intensely fluorodeoxyglucose-avid on positron emission tomography scanning. An initial bronchoscopic exam revealed circumferential narrowing of the entire left main stem bronchus with necrotic and friable material on the medial wall. Neither aspirates from this necrotic area nor bronchial washing were diagnostic. A second bronchoscopy with endobronchial ultrasound evidenced a soft tissue thickening on the medial aspect of the left main stem bronchus underlying the area of necrosis visible endoluminally. Endobronchial ultrasound-guided transbronchial needle aspiration performed in this area revealed multiple fungal elements suggestive of Aspergillus species. CONCLUSION: We describe the first case of invasive aspergillus tracheobronchitis in which the diagnosis was facilitated by the use of endobronchial ultrasound guided trans-bronchial needle aspiration. To the best of our knowledge, we are also presenting the first positron emission tomography scan images of this condition in the literature. We cautiously suggest that endobronchial ultrasound imaging may be a useful tool to evaluate the degree of invasion and the involvement of vascular structures in these patients prior to bronchoscopic manipulation of the affected areas in an effort to avoid potentially fatal hemorrhage.

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