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1.
ACS Synth Biol ; 13(4): 1077-1084, 2024 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-38588591

RESUMO

Uremic toxins (UTs) are microbiota-derived metabolites that accelerate the progression of kidney damage in patients with chronic kidney disease (CKD). One of the major UTs involved in CKD progression is p-cresol-sulfate (PCS), derived from dietary l-tyrosine (l-Tyr). Here, we engineered a probiotic strain of Escherichia coli Nissle 1917, to convert l-Tyr to the nontoxic compound p-coumaric acid via tyrosine ammonia lyase (TAL). First, a small metagenomic library was assessed to identify the TAL with the greatest whole-cell activity. Second, accessory genes implicated in the import of l-Tyr and export of PCA were overexpressed to enhance l-Tyr degradation by 106% and 56%, respectively. Last, random mutagenesis coupled to a novel selection and screening strategy was developed that identified a TAL variant with a 25% increase in whole-cell activity. Taken together, the final strain exhibits a 183% improvement over initial whole-cell activity and provides a promising candidate to degrade l-Tyr mediated PCS accumulation.


Assuntos
Escherichia coli , Insuficiência Renal Crônica , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Toxinas Urêmicas , Mutagênese , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo
2.
Cell Host Microbe ; 32(3): 382-395.e10, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38309259

RESUMO

Methionine is an essential proteinogenic amino acid, but its excess can lead to deleterious effects. Inborn errors of methionine metabolism resulting from loss of function in cystathionine ß-synthase (CBS) cause classic homocystinuria (HCU), which is managed by a methionine-restricted diet. Synthetic biotics are gastrointestinal tract-targeted live biotherapeutics that can be engineered to replicate the benefits of dietary restriction. In this study, we assess whether SYNB1353, an E. coli Nissle 1917 derivative, impacts circulating methionine and homocysteine levels in animals and healthy volunteers. In both mice and nonhuman primates (NHPs), SYNB1353 blunts the appearance of plasma methionine and plasma homocysteine in response to an oral methionine load. A phase 1 clinical study conducted in healthy volunteers subjected to an oral methionine challenge demonstrates that SYNB1353 is well tolerated and blunts plasma methionine by 26%. Overall, SYNB1353 represents a promising approach for methionine reduction with potential utility for the treatment of HCU.


Assuntos
Homocistinúria , Metionina , Humanos , Camundongos , Animais , Metionina/metabolismo , Metionina/uso terapêutico , Voluntários Saudáveis , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Animais de Doenças , Homocistinúria/tratamento farmacológico , Homocistinúria/metabolismo , Racemetionina , Homocisteína/uso terapêutico
3.
Clin Cancer Res ; 29(13): 2435-2444, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37227176

RESUMO

PURPOSE: SYNB1891 is a live, modified strain of the probiotic Escherichia coli Nissle 1917 (EcN) engineered to produce cyclic dinucleotides under hypoxia, leading to STimulator of INterferon Genes (STING) activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. PATIENTS AND METHODS: This first-in-human study (NCT04167137) enrolled participants with refractory advanced cancers to receive repeat intratumoral injections of SYNB1891 either alone or in combination with atezolizumab, with the primary objective of evaluating the safety and tolerability of both regimens. RESULTS: Twenty-four participants received monotherapy across six cohorts, and 8 participants received combination therapy in two cohorts. Five cytokine release syndrome events occurred with monotherapy, including one that met the criteria for dose-limiting toxicity at the highest dose; no other SYNB1891-related serious adverse events occurred, and no SYNB1891-related infections were observed. SYNB1891 was not detected in the blood at 6 or 24 hours after the first intratumoral dose or in tumor tissue 7 days following the first dose. Treatment with SYNB1891 resulted in activation of the STING pathway and target engagement as assessed by upregulation of IFN-stimulated genes, chemokines/cytokines, and T-cell response genes in core biopsies obtained predose and 7 days following the third weekly dose. In addition, a dose-related increase in serum cytokines was observed, as well as stable disease in 4 participants refractory to prior PD-1/L1 antibodies. CONCLUSIONS: Repeat intratumoral injection of SYNB1891 as monotherapy and in combination with atezolizumab was safe and well tolerated, and evidence of STING pathway target engagement was observed.


Assuntos
Escherichia coli , Neoplasias , Humanos , Escherichia coli/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Anticorpos Monoclonais Humanizados , Fatores Imunológicos/uso terapêutico , Citocinas/uso terapêutico
4.
Alcohol Clin Exp Res (Hoboken) ; 47(5): 856-867, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36871955

RESUMO

BACKGROUND AND PURPOSE: Gut bacteria metabolize tryptophan into indoles. Intestinal levels of the tryptophan metabolite indole-3-acetic acid are reduced in patients with alcohol-associated hepatitis. Supplementation of indole-3-acetic acid protects against ethanol-induced liver disease in mice. The aim of this study was to evaluate the effect of engineered bacteria producing indoles as Aryl-hydrocarbon receptor (Ahr) agonists. METHODS: C57BL/6 mice were subjected to chronic-plus-binge ethanol feeding and orally given PBS, control Escherichia coli Nissle 1917 (EcN) or engineered EcN-Ahr. The effects of EcN and EcN-Ahr were also examined in mice lacking Ahr in interleukin 22 (Il22)-producing cells. RESULTS: Through the deletion of endogenous genes trpR and tnaA, coupled with overexpression of a feedback-resistant tryptophan biosynthesis operon, EcN-Ahr were engineered to overproduce tryptophan. Additional engineering allowed conversion of this tryptophan to indoles including indole-3-acetic acid and indole-3-lactic acid. EcN-Ahr ameliorated ethanol-induced liver disease in C57BL/6 mice. EcN-Ahr upregulated intestinal gene expression of Cyp1a1, Nrf2, Il22, Reg3b, and Reg3g, and increased Il22-expressing type 3 innate lymphoid cells. In addition, EcN-Ahr reduced translocation of bacteria to the liver. The beneficial effect of EcN-Ahr was abrogated in mice lacking Ahr expression in Il22-producing immune cells. CONCLUSIONS: Our findings indicate that tryptophan metabolites locally produced by engineered gut bacteria mitigate liver disease via Ahr-mediated activation in intestinal immune cells.

5.
PLoS One ; 18(2): e0280499, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36730255

RESUMO

E. coli Nissle (EcN) is a non-pathogenic probiotic bacterium of the Enterobacteriaceae family that has been used for over a century to promote general gut health. Despite the history of safe usage of EcN, concerns have been raised regarding the presence of the pks gene cluster, encoding the genotoxin colibactin, due to its association with colorectal cancer. Here, we sought to determine the effect of pks island removal on the in vitro and in vivo robustness and activity of EcN and EcN-derived strains. A deletion of the pks island (Δpks) was constructed in wild type and engineered strains of EcN using lambda red recombineering. Mass spectrometric measurement of N-myristoyl-D-asparagine, released during colibactin maturation, confirmed that the pks deletion abrogated colibactin production. Growth curves were comparable between Δpks strains and their isogenic parents, and wild type EcN displayed no competitive advantage to the Δpks strain in mixed culture. Deletion of pks also had no effect on the activity of strains engineered to degrade phenylalanine (SYNB1618 and SYNB1934) or oxalate (SYNB8802). Furthermore, 1:1 mixed dosing of wild type and Δpks EcN in preclinical mouse and nonhuman primate models demonstrated no competitive disadvantage for the Δpks strain with regards to transit time or colonization. Importantly, there was no significant difference on in vivo strain performance between the clinical-stage strain SYNB1934 and its isogenic Δpks variant with regards to recovery of the quantitative strain-specific biomarkers d5- trans-cinnamic acid, and d5-hippuric acid. Taken together, these data support that the pks island is dispensable for Synthetic Biotic fitness and activity in vivo and that its removal from engineered strains of EcN will not have a deleterious effect on strain efficacy.


Assuntos
Proteínas de Escherichia coli , Policetídeos , Camundongos , Animais , Escherichia coli/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Escherichia coli/genética , Policetídeos/metabolismo , Família Multigênica
6.
Mol Syst Biol ; 18(3): e10539, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35253995

RESUMO

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non-human primates, demonstrating the strain's ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.


Assuntos
Hiperoxalúria , Animais , Simulação por Computador , Feminino , Humanos , Hiperoxalúria/etiologia , Hiperoxalúria/urina , Masculino , Camundongos , Oxalatos/metabolismo , Oxalatos/urina
7.
Ann N Y Acad Sci ; 1506(1): 98-117, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34786712

RESUMO

Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.


Assuntos
Congressos como Assunto/tendências , Engenharia Genética/tendências , Terapia Genética/tendências , Relatório de Pesquisa , Biologia Sintética/tendências , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/tendências , Marcação de Genes/métodos , Marcação de Genes/tendências , Engenharia Genética/métodos , Terapia Genética/métodos , Humanos , Células Matadoras Naturais/imunologia , Aprendizado de Máquina/tendências , Biologia Sintética/métodos , Linfócitos T/imunologia
8.
Antibiotics (Basel) ; 10(4)2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33800658

RESUMO

Aspergillus spp. are spore forming molds; a subset of which are clinically relevant to humans and can cause significant morbidity and mortality. A. fumigatus causes chronic infection in patients with chronic lung disease such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). In patients with CF, A. fumigatus infection can lead to allergic disease, such as allergic bronchopulmonary aspergillosis (ABPA) which is associated with high rates of hospitalizations for acute exacerbations and lower lung function. ABPA results from TH2 immune response to Aspergillus antigens produced during hyphal growth, marked by high levels of IgE and eosinophil activation. Clinically, patients with ABPA experience difficulty breathing; exacerbations of disease and are at high risk for bronchiectasis and lung fibrosis. Oral corticosteroids are used to manage aspects of the inflammatory response and antifungal agents are used to reduce fungal burden and lower the exposure to fungal antigens. As the appreciation for the severity of fungal infections has grown, new therapies have emerged that aim to improve treatment and outcomes for patients with CF.

9.
Br J Clin Pharmacol ; 86(4): 723-733, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31696544

RESUMO

AIMS: Oral itraconazole has variable pharmacokinetics and risks of adverse events associated with high plasma exposure. An inhalation formulation of itraconazole (PUR1900) is being developed to treat allergic bronchopulmonary aspergillosis, an allergic inflammatory disease occurring in asthmatics and patients with cystic fibrosis. METHODS: A 3-part, open-label Phase 1 study was conducted to evaluate safety, tolerability and pharmacokinetics of PUR1900. Healthy volunteers (n = 5-6/cohort) received either single (Part 1) or multiple (Part 2) ascending doses of PUR1900 for up to 14 days. In Part 3 stable, adult asthmatics received a single dose of 20 mg PUR1900 or 200 mg of oral Sporanox (itraconazole oral solution) in a 2-period randomized cross-over design. Itraconazole plasma and sputum concentrations were evaluated. RESULTS: None of the adverse events considered as at least possibly related to study treatment were moderate or severe, and none were classed as serious. The most common was the infrequent occurrence of mild cough. Itraconazole plasma exposure increased with increasing doses of PUR1900. After 14 days, PUR1900 resulted in plasma exposure (area under the concentration-time curve up to 24 h) 106- to 400-fold lower across doses tested (10-35 mg) than steady-state exposure reported for oral Sporanox 200 mg. In asthmatics, PUR1900 geometric mean maximum sputum concentrations were 70-fold higher and geometric mean plasma concentrations were 66-fold lower than with oral Sporanox. CONCLUSION: PUR1900 was safe and well-tolerated under the study conditions. Compared to oral dosing, PUR1900 achieved higher lung and lower plasma exposure. The pharmacokinetic profile of PUR1900 suggests the potential to improve upon the efficacy and safety profile observed with oral itraconazole.


Assuntos
Itraconazol , Administração Oral , Adulto , Área Sob a Curva , Estudos de Coortes , Estudos Cross-Over , Voluntários Saudáveis , Humanos , Itraconazol/efeitos adversos
10.
AAPS PharmSciTech ; 20(6): 242, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264190

RESUMO

The utilization of ferrets as a non-clinical model for disease is rapidly increasing within drug development. Many of these models include respiratory diseases that involve targeted drug delivery via nose-only inhalation. While the deposition patterns within other non-clinical models (mice, rats, canines, and non-human primates) have been well studied, the local and regional deposition of aerosols in ferrets has not been well characterized. Therefore, inhalation aerosols were developed, radiolabeled and the radiolabeling methods validated to support SPECT-CT imaging and quantification of regional deposition within ferrets. The studies were conducted with one liquid formulation and one dry powder formulation (two concentrations of dry powder). Additionally, both aerosols were polydisperse and therefore reflect the majority of pharmaceutical aerosols. Overall, the studies showed lung deposition fractions between 5 and 10% with median aerodynamic particle sizes of 2.5 and 2.8 µm. The lung deposition fraction of the liquid aerosol was ~ 9%, nearly double observed in rats with a similarly sized aerosol. Analysis of respiratory tract (oropharynx, laryngopharynx, trachea, bifurcation area, and lung) deposition indicates increased deposition of the liquid aerosol compared to the dry powder aerosol, however, when this analysis was refined to the pulmonary region (trachea, bifurcation, and lung) the deposition was similar between formulations. These data provide the first description of the regional deposition of inhalation aerosols in ferrets with standard nose-only inhalation procedures. These data can be used for calculations of both total and regional doses within ferret inhalation drug delivery.


Assuntos
Aerossóis/farmacocinética , Sistemas de Liberação de Medicamentos , Pulmão/metabolismo , Pós , Administração por Inalação , Animais , Furões , Humanos , Camundongos , Nebulizadores e Vaporizadores , Tamanho da Partícula , Ratos
11.
Br J Clin Pharmacol ; 85(3): 580-589, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30521129

RESUMO

AIMS: Plasma pharmacokinetics permit the assessment of efficacy and safety of inhaled drugs, and possibly their bioequivalence to other inhaled products. Correlating drug product attributes to lung deposited dose is important to achieving equivalence. PUR0200 is a spray-dried formulation of tiotropium that enables more efficient lung delivery than Spiriva® HandiHaler® (HH). The ratio of tiotropium lung-to-oral deposition in PUR0200 was varied to investigate the impact of particle size on tiotropium pharmacokinetics, and the contribution of oral absorption to tiotropium exposure was assessed using charcoal block. METHODS: A seven-period, single-dose, crossover study was performed in healthy subjects. PUR0200 formulations differing in dose and aerodynamic particle size were administered in five periods and Spiriva HH in two periods. In one period, Spiriva HH gastrointestinal absorption was blocked with oral charcoal. Tiotropium plasma concentrations were assessed over 8 h after inhalation. RESULTS: PUR0200 pharmacokinetics were influenced by aerodynamic particle size and the ratio of lung-to-oral deposition, with impactor sized mass (ISM) correlating most strongly with exposure. Formulation PUR0217a (3 µg tiotropium) lung deposition was similar to Spiriva HH (18 µg) with and without charcoal block, but total PUR0200 exposure was lower without charcoal. The Cmax and AUC0-0.5h of Spiriva HH with and without charcoal block were bioequivalent; however, Spiriva HH AUC0-8h was lower when gastrointestinal absorption was inhibited with oral charcoal administration. CONCLUSIONS: Pharmacokinetic bioequivalence indicative of lung deposition and efficacy can be achieved by matching the reference product ISM. Due to reduced oral deposition and more efficient lung delivery, PUR0200 results in a lower AUC0-t than Spiriva HH due to reduced absorption of drug from the gastrointestinal tract.


Assuntos
Broncodilatadores/farmacocinética , Absorção Gastrointestinal , Mucosa Bucal/metabolismo , Brometo de Tiotrópio/farmacocinética , Administração por Inalação , Adulto , Área Sob a Curva , Broncodilatadores/administração & dosagem , Broncodilatadores/química , Estudos Cross-Over , Inaladores de Pó Seco , Feminino , Voluntários Saudáveis , Humanos , Masculino , Tamanho da Partícula , Projetos Piloto , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Equivalência Terapêutica , Brometo de Tiotrópio/administração & dosagem , Brometo de Tiotrópio/química , Adulto Jovem
12.
Pulm Pharmacol Ther ; 53: 86-99, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30359782

RESUMO

Tobacco smoke-induced lung inflammation in patients with chronic obstructive pulmonary disease (COPD) worsens with disease progression and acute exacerbations caused by respiratory infections. Chronic therapies to manage COPD center on bronchodilators to improve lung function and inhaled corticosteroids (ICS) to help reduce the risk of exacerbations. Novel therapies are needed that reduce the underlying inflammation associated with COPD and the inflammation resulting from respiratory infections that worsen disease. The lung is lined with airway surface liquid (ASL), a rheologically active material that provides an innate defense for the airway against inhaled particulate and is continuously cleared from the airways by mucociliary clearance. The rheological properties of the ASL can be altered by changes in airway hydration and by cations, such as calcium, that interact with electronegative glycoproteins. The effect of inhaled salts on inflammation resulting from tobacco smoke exposure was studied to determine if cations could be used to alter the properties of the ASL and reduce inflammation. Inhaled calcium salts, but not sodium or magnesium salts, reduced cellular inflammation and key chemokines and cytokines that were induced by tobacco smoke exposure. Similar anti-inflammatory effects of calcium salts were observed using in vitro cultures of human monocyte derived macrophages and human bronchial epithelial cells. The data suggest that inhaled calcium salts may act broadly on both biophysical and biological pathways to reduce pulmonary inflammation.


Assuntos
Cálcio/farmacologia , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Fumaça/efeitos adversos , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/patologia , Cálcio/química , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Humanos , Inflamação/etiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Pneumonia/etiologia , Pneumonia/prevenção & controle , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Sais , Nicotiana/toxicidade
13.
Br J Clin Pharmacol ; 84(9): 2097-2105, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29790581

RESUMO

AIMS: PUR0200 is a tiotropium bromide formulation engineered with the iSPERSE dry powder delivery technology. PUR0200 is being developed as a bioequivalent alternative to tiotropium bromide, delivered using Spiriva® HandiHaler® (HH). We investigated the bronchodilator effects, pharmacokinetics and safety of PUR0200 in patients with chronic obstructive pulmonary disease (COPD). METHODS: This was a randomized, placebo-controlled, crossover study using different PUR0200 doses and the comparator tiotropium HH. In vitro aerodynamic particle size distribution (aPSD) characterization of PUR0200 and tiotropium HH are presented. The main endpoints included forced expiratory volume in 1 s (FEV1 ) trough and (0-24 h) and pharmacokinetic parameters. RESULTS: The increased fine-particle fraction of PUR0200 demonstrated by testing using the next-generation impactor increased the proportion of drug available for lung deposition compared with the tiotropium HH. There was a numerical dose-response effect for PUR0200 on FEV1 , with 3 µg demonstrating a lower effect than higher doses. The placebo-adjusted mean (95% confidence interval) increases from baseline at 24 h postdose were 150 ml (100-200), 210 ml (160-270) and 200 ml (140-250) for 3 µg, 6 µg and 9 µg doses of PUR0200, respectively. Tiotropium HH (18 µg) caused a mean 169 ml (standard deviation 157ml) improvement in trough FEV1 , which was not significantly different to the PUR0200 effects at any of the tested doses. CONCLUSIONS: PUR0200 treatment caused bronchodilation in COPD patients that was similar in magnitude to that caused by tiotropium HH. This enabled a similar clinical effect on lung function to be achieved with PUR0200 using a lower metered dose of tiotropium compared with tiotropium HH.


Assuntos
Broncodilatadores/farmacocinética , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Brometo de Tiotrópio/farmacocinética , Administração por Inalação , Idoso , Broncodilatadores/administração & dosagem , Broncodilatadores/efeitos adversos , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Inaladores de Pó Seco , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Espirometria , Equivalência Terapêutica , Brometo de Tiotrópio/administração & dosagem , Brometo de Tiotrópio/efeitos adversos , Resultado do Tratamento
14.
Am J Respir Cell Mol Biol ; 52(6): 728-37, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25338285

RESUMO

Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have demonstrated impaired bacterial phagocytosis and disordered cytokine secretion, which are calcium-dependent processes. We determined how calcium moderates the macrophage response to nontypeable Haemophilus influenzae (NTHI). We hypothesized that augmenting extracellular calcium during bacterial challenge would restore bacterial phagocytosis and cytokine secretion in monocyte-derived macrophages (MDMs) from subjects with COPD. We further determined whether restoration of pattern recognition and scavenger receptors correlated with the calcium-induced improvements in macrophage function. Monocytes were purified from whole blood from healthy control subjects (n = 20) and patients with moderate to severe COPD (n = 35), and cultured in suspension with granulocyte macrophage colony-stimulating factor to generate MDMs. The MDMs were incubated with fluorescently labeled NTHI with and without calcium lactate and calcium channel inhibitors. Phagocytosis efficiency was evaluated by flow cytometry. Supernatants were assayed for cytokines using bead array technology. Cell surface receptor expression was assayed by multicolor flow cytometry. Extracellular calcium significantly improved phagocytosis and cytokine secretion (IL-8, TNF-α, and macrophage inflammatory protein [MIP]-1α, and -1ß) in COPD MDMs. NTHI challenge led to statistically significant reductions in CD16 (FcγRIII), and extracellular calcium up-regulated both CD16 and macrophage receptor with collagenous structure (MARCO). Specific calcium channel inhibitors abrogated calcium-mediated MARCO up-regulation and cytokine secretion. Extracellular calcium improved phagocytosis, restored innate cytokine secretion, and increased cell surface expression of bacterial recognition receptors, CD16 and MARCO. These observations support the therapeutic use of calcium to improve macrophage function in COPD to decrease exacerbations and chronic bacterial infection.


Assuntos
Cálcio/farmacologia , Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Macrófagos Alveolares/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Cultivadas , Citocinas/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunidade Celular , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Masculino , Pessoa de Meia-Idade , Fagocitose , Doença Pulmonar Obstrutiva Crônica/microbiologia , Receptores de IgG/metabolismo
15.
Immunity ; 35(2): 182-93, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21802320

RESUMO

Antigen presentation and microbial killing are critical arms of host defense that depend upon cargo trafficking into lysosomes. Yet, the molecular regulators of traffic into lysosomes are only partly understood. Here, using a lysosome-dependent immunological screen of a trafficking shRNA library, we identified the Arf-like GTPase Arl8b as a critical regulator of cargo delivery to lysosomes. Homotypic fusion and vacuole protein sorting (HOPS) complex members were identified as effectors of Arl8b and were dependent on Arl8b for recruitment to lysosomes, suggesting that Arl8b-HOPS plays a general role in directing traffic to lysosomes. Moreover, the formation of CD1 antigen-presenting complexes in lysosomes, their delivery to the plasma membrane, and phagosome-lysosome fusion were all markedly impaired in Arl8b silenced cells resulting in corresponding defects in T cell activation and microbial killing. Together, these results define Arl8b as a key regulator of lysosomal cellular and immunological functions.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Antígenos/metabolismo , Lisossomos/metabolismo , Células T Matadoras Naturais/metabolismo , Proteolipídeos/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/imunologia , Apresentação de Antígeno/genética , Antígenos/imunologia , Antígenos CD1d/metabolismo , Citocinas/genética , Citocinas/metabolismo , Citotoxicidade Imunológica/genética , Células HeLa , Humanos , Ativação Linfocitária/genética , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/patologia , Ligação Proteica/genética , Transporte Proteico/genética , Proteolipídeos/imunologia , RNA Interferente Pequeno/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
16.
Proc Natl Acad Sci U S A ; 105(32): 11281-6, 2008 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-18685099

RESUMO

Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD1/imunologia , Células Dendríticas/imunologia , Lipídeos/imunologia , Mycobacterium tuberculosis/imunologia , Peptídeos/imunologia , Tuberculose/imunologia , Transporte Biológico Ativo/imunologia , Membrana Celular/imunologia , Células Cultivadas , Células Dendríticas/microbiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Cinética , Fagocitose/imunologia , Fagossomos/imunologia , Linfócitos T/imunologia
17.
Eur J Immunol ; 38(8): 2351-9, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18624350

RESUMO

Lipid antigens are presented to T cells by the non-polymorphic MHC class I-related CD1 molecules. Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum (ER)-resident chaperone that has been shown to lipidate the group 2 CD1 molecule CD1d and thus to regulate its function. We now report that MTP also regulates the function of group 1 CD1 molecules CD1a, CD1b, and CD1c. Pharmacological inhibition of MTP in monocyte-derived dendritic cells and lymphoblastoid B cell lines transfected with group 1 CD1 resulted in a substantial decrease in endogenous self lipid antigen presentation to several CD1-restricted T cell lines. Silencing MTP expression in CD1c-transfected HeLa cells similarly resulted in decreased self reactivity. Unexpectedly, inhibition of ER-resident MTP, which was confirmed by confocal microscopy, also markedly decreased presentation of exogenous, endosomally loaded, mycobacterial lipid antigens by CD1a and CD1c to T cells. Thus, these studies indicate that MTP, despite its ER localization, regulates endogenous as well as exogenous lipid antigen presentation, and suggest a broad role for MTP in the regulation of CD1 antigen presentation.


Assuntos
Apresentação de Antígeno , Antígenos CD1/fisiologia , Proteínas de Transporte/fisiologia , Glicoproteínas/fisiologia , Células HeLa , Humanos
18.
Immunology ; 125(3): 289-301, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18445008

RESUMO

Dendritic cells (DCs) are highly potent antigen-presenting cells (APCs) and play a vital role in stimulating naïve T cells. Treatment of human blood monocytes with the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 stimulates them to develop into immature dendritic cells (iDCs) in vitro. DCs generated by this pathway have a high capacity to prime and activate resting T cells and prominently express CD1 antigen-presenting molecules on the cell surface. The presence of human serum during the differentiation of iDCs from monocytes inhibits the expression of CD1a, CD1b and CD1c, but not CD1d. Correspondingly, T cells that are restricted by CD1c showed poor responses to DCs that were generated in the presence of human serum, while the responses of CD1d-restricted T cells were enhanced. We chemically fractionated human serum to isolate the bioactive factors that modulate surface expression of CD1 proteins during monocyte to DC differentiation. The human serum components that affected CD1 expression partitioned with polar organic soluble fractions. Lysophosphatidic acid and cardiolipin were identified as lipids present in normal human serum that potently modulate CD1 expression. Control of CD1 expression was mediated at the level of gene transcription and correlated with activation of the peroxisome proliferator-activated receptor (PPAR) nuclear hormone receptors. These findings indicate that the ability of human DCs to present lipid antigens to T cells through expression of CD1 molecules is sensitively regulated by lysophosphatidic acid and cardiolipin in serum, which are ligands that can activate PPAR transcription factors.


Assuntos
Antígenos CD1/metabolismo , Células Dendríticas/imunologia , Lipídeos/imunologia , Apresentação de Antígeno/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Expressão Gênica , Humanos , Ativação Linfocitária/imunologia , Receptores Ativados por Proliferador de Peroxissomo/biossíntese , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/imunologia , Soro/imunologia , Subpopulações de Linfócitos T/imunologia
20.
Curr Opin Immunol ; 17(1): 88-94, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15653316

RESUMO

The CD1 antigen presentation system presents lipid antigens to effector T cells, which have diverse roles in antimicrobial responses, antitumor immunity and in regulating the balance between tolerance and autoimmunity. The trafficking of CD1 molecules and lipid antigens facilitates their intersection and binding in specific intracellular compartments. Recent studies have now identified unexpected accessory molecules that are critical to CD1 assembly and lipid loading. The atomic structures of CD1-antigen complexes have defined both the orientation of polar headgroups between the alpha1 and alpha2 helices of CD1 and the manner in which distinct CD1 isoforms bind a range of lipids that have different lengths and numbers of hydrocarbon chains.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD1/imunologia , Lipídeos/imunologia , Animais , Antígenos/química , Antígenos/imunologia , Antígenos CD1/química , Humanos , Lipídeos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Estrutura Terciária de Proteína , Linfócitos T/imunologia
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