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2.
Cell ; 179(2): 417-431.e19, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585081

RESUMO

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

3.
Pharmacol Ther ; 190: 202-236, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29842917

RESUMO

Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects mediated by these proteases in inflammatory/auto-immune disorders. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome (PLS). The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Catepsina C/antagonistas & inibidores , Inflamação/tratamento farmacológico , Animais , Doenças Autoimunes/fisiopatologia , Catepsina C/metabolismo , Desenvolvimento de Medicamentos/métodos , Humanos , Inflamação/fisiopatologia , Doença de Papillon-Lefevre/tratamento farmacológico , Doença de Papillon-Lefevre/fisiopatologia , Serina Proteases/metabolismo
4.
J Hosp Med ; 12(2): 104-108, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28182807

RESUMO

The approach to clinical conundrums by an expert clinician is revealed through the presentation of an actual patient's case in an approach typical of a morning report. Similarly to patient care, sequential pieces of information are provided to the clinician, who is unfamiliar with the case. The focus is on the thought processes of both the clinical team caring for the patient and the discussant.


Assuntos
Anti-Inflamatórios/administração & dosagem , Diagnóstico Diferencial , Mastocitose Sistêmica , Prednisona/administração & dosagem , Acetatos/administração & dosagem , Idoso , Humanos , Hipotensão , Antagonistas de Leucotrienos/administração & dosagem , Masculino , Mastocitose Sistêmica/tratamento farmacológico , Mastocitose Sistêmica/patologia , Quinolinas/administração & dosagem , Taquicardia
5.
Nat Genet ; 48(12): 1564-1569, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27749843

RESUMO

Elevated basal serum tryptase levels are present in 4-6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase-encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.


Assuntos
Dor Crônica/genética , Doenças do Tecido Conjuntivo/genética , Variações do Número de Cópias de DNA/genética , Disautonomia Familiar/genética , Gastroenteropatias/genética , Prurido/genética , Dermatopatias/genética , Triptases/sangue , Triptases/genética , Adolescente , Adulto , Idoso , Criança , Dor Crônica/sangue , Dor Crônica/enzimologia , Doenças do Tecido Conjuntivo/sangue , Doenças do Tecido Conjuntivo/enzimologia , Disautonomia Familiar/sangue , Disautonomia Familiar/enzimologia , Feminino , Gastroenteropatias/sangue , Gastroenteropatias/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Prurido/sangue , Prurido/enzimologia , Dermatopatias/sangue , Dermatopatias/enzimologia , Adulto Jovem
6.
PLoS One ; 11(10): e0164501, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27716790

RESUMO

Host-derived proteases can augment or help to clear infections. This dichotomy is exemplified by cathepsin L (CTSL), which helps Hendra virus and SARS coronavirus to invade cells, but is essential for survival in mice with mycoplasma pneumonia. The present study tested the hypothesis that CTSL protects mice from serious consequences of infection by the orthomyxovirus influenza A, which is thought to be activated by host-supplied proteases other than CTSL. Ctsl-/- mice infected with influenza A/Puerto Rico/8/34(H1N1) had larger lung viral loads and higher mortality than infected Ctsl+/+ mice. Lung inflammation in surviving infected mice peaked 14 days after initial infection, accompanied marked focal distal airway bronchiolization and epithelial metaplasia followed by desquamation and fibrotic interstitial remodeling, and persisted for at least 6 weeks. Most deaths occurred during the second week of infection in both groups of mice. In contrast to mycoplasma pneumonia, infiltrating cells were predominantly mononuclear rather than polymorphonuclear. The histopathology of lung inflammation and remodeling in survivors was similar in Ctsl-/- and Ctsl+/+ mice, although Ctsl+/+ mice cleared immunoreactive virus sooner. Furthermore, Ctsl-/- mice had profound deficits in CD4+ lymphocytes before and after infection and weaker production of pathogen-specific IgG. Thus, CTSL appears to support innate as well as adaptive responses, which confer a survival advantage on mice infected with the orthomyxovirus influenza A.


Assuntos
Catepsina L/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Feminino , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/tratamento farmacológico , Pneumonia/virologia
7.
Transplantation ; 100(10): 2090-8, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27077597

RESUMO

BACKGROUND: Acute cellular rejection is a major cause of morbidity after lung transplantation. Because regulatory T (Treg) cells limit rejection of solid organs, we hypothesized that donor-reactive Treg increase after transplantation with development of partial tolerance and decrease relative to conventional CD4 (Tconv) and CD8 T cells during acute cellular rejection. METHODS: To test these hypotheses, we prospectively collected 177 peripheral blood mononuclear cell specimens from 39 lung transplant recipients at the time of transplantation and during bronchoscopic assessments for acute cellular rejection. We quantified the proportion of Treg, CD4 Tconv, and CD8 T cells proliferating in response to donor-derived, stimulated B cells. We used generalized estimating equation-adjusted regression to compare donor-reactive T cell frequencies with acute cellular rejection pathology. RESULTS: An average of 16.5 ± 9.0% of pretransplantation peripheral blood mononuclear cell Treg cell were donor-reactive, compared with 3.8% ± 2.9% of CD4 Tconv and 3.4 ± 2.6% of CD8 T cells. These values were largely unchanged after transplantation. Donor-reactive CD4 Tconv and CD8 T cell frequencies both increased 1.5-fold (95% confidence interval [95% CI], 1.3-1.6; P < 0.001 and 95% CI, 1.2-1.6; P = 0.007, respectively) during grade A2 rejection compared with no rejection. Surprisingly, donor-reactive Treg frequencies increased by 1.7-fold (95% CI, 1.4-1.8; P < 0.001). CONCLUSIONS: Contrary to prediction, overall proportions of donor-reactive Treg cells are similar before and after transplantation and increase during grade A2 rejection. This suggests how A2 rejection can be self-limiting. The observed increases over high baseline proportions in donor-reactive Treg were insufficient to prevent acute lung allograft rejection.


Assuntos
Rejeição de Enxerto/imunologia , Transplante de Pulmão/efeitos adversos , Linfócitos T Reguladores/imunologia , Doadores de Tecidos , Doença Aguda , Idoso , Feminino , Teste de Histocompatibilidade , Humanos , Masculino , Pessoa de Meia-Idade , Transplante Homólogo
8.
Eur J Pharmacol ; 778: 44-55, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25958181

RESUMO

Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human ß-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the properties and patterns of expression of proteases expressed in human mast cell subsets, and in humans versus other mammals. These considerations are influencing prioritization of specific protease targets for therapeutic inhibition, as well as options of pre-clinical models, disease indications, and choice of topical versus systemic routes of inhibitor administration.


Assuntos
Mastócitos/enzimologia , Terapia de Alvo Molecular/métodos , Peptídeo Hidrolases/metabolismo , Animais , Humanos , Mastócitos/efeitos dos fármacos , Peptídeo Hidrolases/química
9.
PLoS One ; 10(10): e0141169, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26485396

RESUMO

Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and ß-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.


Assuntos
Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Serina Endopeptidases/química , Inibidores de Serino Proteinase/farmacologia , Triptases/antagonistas & inibidores , Aprotinina/farmacologia , Brônquios/citologia , Brônquios/enzimologia , Domínio Catalítico , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Gabexato/análogos & derivados , Gabexato/farmacologia , Humanos
10.
PLoS One ; 10(5): e0125797, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25938594

RESUMO

Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi(+/+) and Dppi(-/-) mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi(-/-) versus Dppi(+/+) lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Peptídeo Hidrolases/metabolismo , Pneumonia/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Catepsina C/genética , Modelos Animais de Doenças , Expressão Gênica , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Elastase de Leucócito/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Mycoplasma pulmonis/enzimologia , Especificidade de Órgãos/genética , Pneumonia/genética , Pneumonia/microbiologia , Pneumonia/patologia , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/metabolismo , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/patologia , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/metabolismo
11.
PLoS One ; 10(3): e0118513, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25789765

RESUMO

Mast cells (MC) and myeloid dendritic cells (DC) act proximally in detecting and processing antigens and immune insults. We sought to understand their comparative dynamic behavior with respect to the airway epithelium in the steady state and in response to an allergic stimulus in mouse trachea. We devised methods to label MC in living trachea and to demonstrate that MC and DC occupy distinct layers of the tracheal mucosa, with DC being closer to the lumen. DC numbers doubled after allergen challenge, but MC numbers remained stable. MC and DC migrated minimally in either steady state or allergen-challenge conditions, and their interactions with one another appeared to be stochastic and relatively infrequent. While DC, unlike MC, exhibited probing behaviors involving dendrites, these projections did not cross the epithelium into the airway lumen. MC typically were located too far from the epithelial surface to contact the tracheal lumen. However, MC had protrusions toward and into blood vessels, likely to load with IgE. Thus, DC and MC occupy distinct niches and engage in sessile surveillance in the mouse trachea. Little or no access of these cell types to the airway lumen suggests that trans-epithelial transport of proteins in the steady state would be required for them to access luminal antigens.


Assuntos
Alérgenos/imunologia , Vasos Sanguíneos/imunologia , Vasos Sanguíneos/patologia , Extensões da Superfície Celular/imunologia , Mastócitos/citologia , Mastócitos/imunologia , Traqueia/imunologia , Animais , Movimento Celular , Células Dendríticas/imunologia , Imagem Tridimensional , Imunoglobulina E/imunologia , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Reprodutibilidade dos Testes , Coloração e Rotulagem
12.
Respir Res ; 15: 95, 2014 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25115556

RESUMO

Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit+/+) and mast cell-deficient (KitW-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient KitW-sh/W-sh mice did not differ from those of Kit+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected KitW-sh/W-sh lungs. However, lung injury in BMCMC-injected KitW-sh/W-sh and Kit+/+ mice did not differ in globally mast cell-deficient, uninjected KitW-sh/W-sh mice or in wild-type Kit+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.


Assuntos
Lesão Pulmonar Aguda/patologia , Modelos Animais de Doenças , Mastócitos/patologia , Disfunção Primária do Enxerto/patologia , Lesão Pulmonar Aguda/imunologia , Animais , Células Cultivadas , Humanos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Disfunção Primária do Enxerto/imunologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia
13.
Biochem Biophys Res Commun ; 450(1): 818-823, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24955853

RESUMO

Prior work established that a deficiency in the cysteine protease dipeptidyl peptidase I (DPPI) improves survival following polymicrobial septic peritonitis. To test whether DPPI regulates survival from severe lung infections, DPPI(-/-) mice were studied in a Klebsiella pneumoniae lung infection model, finding that survival in DPPI(-/-) mice is significantly better than in DPPI(+/+) mice 8d after infection. DPPI(-/-) mice have significantly fewer bacteria in the lung than infected DPPI(+/+) mice, but no difference in lung histopathology, lung injury, or cytokine levels. To explore mechanisms of enhanced bacterial clearance in DPPI(-/-) mice, we examined the status of pulmonary collectins, finding that levels of surfactant protein D, but not of surfactant protein A, are higher in DPPI(-/-) than in DPPI(+/+) BAL fluid, and that DPPI(-/-) BAL fluid aggregate bacteria more effectively than control BAL fluid. Sequencing of the amino terminus of surfactant protein D revealed two or eight additional amino acids in surfactant protein D isolated from DPPI(-/-) mice, suggesting processing by DPPI. These results establish that DPPI is a major determinant of survival following Klebsiella pneumoniae lung infection and suggest that the survival disadvantage in DPPI(+/+) mice is in part due to processing of surfactant protein D by DPPI.


Assuntos
Catepsina C/metabolismo , Infecções por Klebsiella/metabolismo , Klebsiella pneumoniae/metabolismo , Pulmão/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Taxa de Sobrevida , Animais , Catepsina C/genética , Infecções por Klebsiella/microbiologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Tecidual
14.
Infect Immun ; 81(10): 3515-26, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23690397

RESUMO

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function. Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings reveal that malarial-parasite-infected mice, like humans, develop L-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of L-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with L-arginine or L-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with L-citrulline also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing L-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans.


Assuntos
Arginina/deficiência , Bacteriemia/imunologia , Intestinos/citologia , Malária/complicações , Mastócitos/fisiologia , Salmonelose Animal/microbiologia , Animais , Bacteriemia/microbiologia , Citrulina , Feminino , Intestinos/imunologia , Intestinos/patologia , Camundongos , Permeabilidade , Plasmodium yoelii , Salmonelose Animal/patologia
15.
Am J Respir Cell Mol Biol ; 49(3): 437-44, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23600672

RESUMO

Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.


Assuntos
Catepsina L/imunologia , Expressão Gênica/imunologia , Pulmão/enzimologia , Linfangiogênese/imunologia , Vasos Linfáticos/imunologia , Infecções por Mycoplasma/enzimologia , Doença Aguda , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Carga Bacteriana , Catepsina L/deficiência , Catepsina L/genética , Doença Crônica , Interferon gama/sangue , Interferon gama/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/mortalidade , Mycoplasma pulmonis/crescimento & desenvolvimento , Índice de Gravidade de Doença , Análise de Sobrevida
16.
J Biol Chem ; 288(15): 10588-98, 2013 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-23447538

RESUMO

Human and mouse marapsins (Prss27) are serine proteases preferentially expressed by stratified squamous epithelia. However, mouse marapsin contains a transmembrane anchor absent from the human enzyme. To gain insights into physical forms, activities, inhibition, and roles in epithelial differentiation, we traced tail loss in human marapsin to a nonsense mutation in an ancestral ape, compared substrate preferences of mouse and human marapsins with those of the epithelial peptidase prostasin, designed a selective substrate and inhibitor, and generated Prss27-null mice. Phylogenetic analysis predicts that most marapsins are transmembrane proteins. However, nonsense mutations caused membrane anchor loss in three clades: human/bonobo/chimpanzee, guinea pig/degu/tuco-tuco/mole rat, and cattle/yak. Most marapsin-related proteases, including prostasins, are type I transmembrane proteins, but the closest relatives (prosemins) are not. Soluble mouse and human marapsins are tryptic with subsite preferences distinct from those of prostasin, lack general proteinase activity, and unlike prostasins resist antiproteases, including leupeptin, aprotinin, serpins, and α2-macroglobulin, suggesting the presence of non-canonical active sites. Prss27-null mice develop normally in barrier conditions and are fertile without overt epithelial defects, indicating that marapsin does not play critical, non-redundant roles in development, reproduction, or epithelial differentiation. In conclusion, marapsins are conserved, inhibitor-resistant, tryptic peptidases. Although marapsins are type I transmembrane proteins in their typical form, they mutated independently into anchorless forms in several mammalian clades, including one involving humans. Similar pathways appear to have been traversed by prosemins and tryptases, suggesting that mutational tail loss is an important means of evolving new functions of tryptic serine proteases from transmembrane ancestors.


Assuntos
Evolução Molecular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Cobaias , Humanos , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Mutantes , Ratos-Toupeira , Mutação , Pan paniscus , Pan troglodytes , Inibidores de Proteases/farmacologia , Ratos , Solubilidade , Especificidade da Espécie
17.
Am J Respir Crit Care Med ; 187(4): 417-23, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23239157

RESUMO

RATIONALE: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk. OBJECTIVES: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality. METHODS: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0-2 "E-score" and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively. MEASUREMENTS AND MAIN RESULTS: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1-3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11-2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01). CONCLUSIONS: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.


Assuntos
Brônquios/patologia , Bronquiolite Obliterante/patologia , Transplante de Pulmão/patologia , Linfócitos/patologia , Biópsia , Bronquite/patologia , Broncoscopia/métodos , Feminino , Rejeição de Enxerto/patologia , Humanos , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fatores de Risco , Índice de Gravidade de Doença , Síndrome
18.
Am J Physiol Lung Cell Mol Physiol ; 303(2): L97-106, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22582115

RESUMO

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.


Assuntos
Membrana Celular/enzimologia , Células Epiteliais/enzimologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Aprotinina/química , Aprotinina/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Polaridade Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Oligopeptídeos/química , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/imunologia , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Especificidade por Substrato
19.
J Clin Invest ; 122(2): 748-58, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22232213

RESUMO

Allergic asthma is the most common form of asthma, affecting more than 10 million Americans. Although it is clear that mast cells have a key role in the pathogenesis of allergic asthma, the mechanisms by which they regulate airway narrowing in vivo remain to be elucidated. Here we report that mice lacking αvß6 integrin are protected from exaggerated airway narrowing in a model of allergic asthma. Expression microarrays of the airway epithelium revealed mast cell proteases among the most prominent differentially expressed genes, with expression of mouse mast cell protease 1 (mMCP-1) induced by allergen challenge in WT mice and expression of mMCP-4, -5, and -6 increased at baseline in ß6-deficient mice. These findings were most likely explained by loss of TGF-ß activation, since the epithelial integrin αvß6 is a critical activator of latent TGF-ß, and in vitro-differentiated mast cells showed TGF-ß-dependent expression of mMCP-1 and suppression of mMCP-4 and -6. In vitro, mMCP-1 increased contractility of murine tracheal rings, an effect that depended on intact airway epithelium, whereas mMCP-4 inhibited IL-13-induced epithelial-independent enhancement of contractility. These results suggest that intraepithelial activation of TGF-ß by the αvß6 integrin regulates airway responsiveness by modulating mast cell protease expression and that these proteases and their proteolytic substrates could be novel targets for improved treatment of allergic asthma.


Assuntos
Antígenos de Neoplasias/imunologia , Hiper-Reatividade Brônquica/imunologia , Epitélio/imunologia , Integrinas/imunologia , Mastócitos/imunologia , Animais , Antígenos de Neoplasias/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Células Cultivadas , Quimases/genética , Quimases/imunologia , Perfilação da Expressão Gênica , Humanos , Integrinas/genética , Interleucina-13/imunologia , Pulmão/anatomia & histologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/fisiologia , Mastócitos/citologia , Mastócitos/enzimologia , Mastócitos/fisiologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Traqueia/anatomia & histologia , Traqueia/fisiologia , Fator de Crescimento Transformador beta/imunologia
20.
J Leukoc Biol ; 91(2): 275-84, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22075928

RESUMO

Airway diseases often feature persistent neutrophilic inflammation and infection. In cystic fibrosis bronchitis, for example, Pseudomonas aeruginosa is isolated frequently. Previously, this laboratory revealed that neutrophils become major sources of histamine in mice with tracheobronchitis caused by the wall-less bacterium Mycoplasma pulmonis. To test the hypothesis that more-broadly pathogenic P. aeruginosa (which expresses cell wall-associated LPS and novel toxins) has similar effects, we incubated naïve mouse neutrophils with two strains of P. aeruginosa. Strain PAO1 greatly increased neutrophil histamine content and secretion, whereas strain PA103 depressed histamine production by killing neutrophils. The histamine-stimulating capacity of PAO1, but not PA103-mediated toxicity, persisted in heat-killed organisms. In PAO1-infected mice, lung and neutrophil histamine content increased. However, PAO1 did not alter production by mast cells (classical histamine reservoirs), which also resisted PA103 toxicity. To explore mechanisms of neutrophil-selective induction, we measured changes in mRNA encoding histidine decarboxylase (rate-limiting for histamine synthesis), probed involvement of endotoxin-TLR pathways in Myd88-deficient neutrophils, and examined contributions of pyocyanin and exotoxins. Results revealed that PAO1 increased histamine production by up-regulating histidine decarboxylase mRNA via pathways largely independent of TLR, pyocyanin, and type III secretion system exotoxins. PAO1 also increased histidine decarboxylase mRNA in neutrophils purified from infected lung. Stimulation required direct contact with neutrophils and was blocked by phagocytosis inhibitor cytochalasin D. In summary, Pseudomonas-augmented histamine production by neutrophils is strain-dependent in vitro and likely mediated by up-regulation of histidine decarboxylase. These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine.


Assuntos
Liberação de Histamina , Histamina/biossíntese , Neutrófilos/metabolismo , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/fisiologia , ADP Ribose Transferases/farmacologia , Animais , Apoptose , Toxinas Bacterianas/farmacologia , Biofilmes , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Meios de Cultivo Condicionados/farmacologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Mastócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Neutrófilos/microbiologia , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Piocianina/farmacologia , Especificidade da Espécie , Organismos Livres de Patógenos Específicos
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