RESUMO
Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant "hot spot 1" mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Candida albicans , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Farmacorresistência Fúngica/genética , Fluconazol/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Especificidade por SubstratoRESUMO
PENICILLIUM MARNEFFEI: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H+ antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2, and PmMDR3) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. PmMDR1-expressing strain showed resistance to the broadest range of drugs, followed by the PmMDR3, and PmMDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3, may be able to serve as multidrug efflux pumps.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Micoses/metabolismo , Talaromyces/metabolismo , Triazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Anfotericina B/uso terapêutico , Sudeste Asiático/epidemiologia , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Humanos , Hospedeiro Imunocomprometido , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , Micoses/epidemiologia , Micoses/microbiologia , Filogenia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Talaromyces/efeitos dos fármacos , Transcriptoma , Triazóis/uso terapêuticoRESUMO
The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antifúngicos/farmacologia , Candida albicans/genética , Proteínas Fúngicas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tacrolimo/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Candida albicans/efeitos dos fármacos , Depsipeptídeos/farmacologia , Farmacorresistência Fúngica/genética , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
BACKGROUND: Although development of immune checkpoint inhibitors and various molecular target agents has extended overall survival time (OS) in advanced non-small cell lung cancer (NSCLC), a complete cure remains rare. We aimed to identify features and treatment modalities of complete remission (CR) cases in stages III and IV NSCLC by analyzing long-term survivors whose OS exceeded 3 years. METHODS: From our hospital database, 1,699 patients, registered as lung cancer between 1st Mar 2004 and 30th Apr 2011, were retrospectively examined. Stage III or IV histologically or cytologically confirmed NSCLC patients with chemotherapy initiated during this period were enrolled. A Cox proportion hazards regression model was used. Data collection was closed on 13th Feb 2017. RESULTS: There were 164 stage III and 279 stage IV patients, including 37 (22.6%) and 51 (18.3%) long-term survivors and 12 (7.3%) and 5 (1.8%) CR patients, respectively. The long-term survivors were divided into three groups: 3 ≤ OS < 5 years, 5 years ≤ OS with tumor, and 5 years ≤ OS without tumor (CR). The median OS of these groups were 1,405, 2,238, and 2,876 days in stage III and 1,368, 2,503, and 2,643 days in stage IV, respectively. The mean chemotherapy cycle numbers were 16, 20, and 10 in stage III and 24, 25, and 5 in stage IV, respectively. In the stage III CR group, all patients received chemoradiation, all oligometastases were controlled by radiation, and none had brain metastases. Compared with non-CR patients, the stage IV CR patients had smaller primary tumors and fewer metastases, which were independent prognostic factors for OS among long-term survivors. The 80% stage IV CR patients received radiation or surgery for controlling primary tumors, and the surgery rate for oligometastases was high. Pathological findings in the stage IV CR patients revealed that numerous inflammatory cells existed around and inside resected lung and brain tumors, indicating strong immune response. CONCLUSIONS: Multiple line chemotherapies with primary and oligometastatic controls by surgery and/or radiation might achieve cure in certain advanced NSCLC. Cure strategies must be changed according to stage III or IV. This study was retrospectively registered on 16th Jun 2019 in UMIN Clinical Trials Registry (number UMIN000037078).
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Estadiamento de Neoplasias/tendências , Indução de Remissão/métodos , Estudos RetrospectivosRESUMO
The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Candida/genética , Candida/metabolismo , Antifúngicos/farmacologia , Clonagem Molecular , Farmacorresistência Fúngica Múltipla , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.
Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Farmacorresistência Fúngica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Penicillium/genética , Penicillium/metabolismo , Sudeste Asiático , Clonagem Molecular , Expressão Gênica , Genoma Fúngico , Humanos , Penicillium/isolamento & purificação , Transporte Proteico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaAssuntos
Asma/complicações , COVID-19/complicações , Corticosteroides/administração & dosagem , Asma/diagnóstico , Asma/patologia , Asma/terapia , COVID-19/diagnóstico , COVID-19/patologia , COVID-19/terapia , Terapia Combinada , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade , Exacerbação dos SintomasRESUMO
BACKGROUND: Protectin D1 (PD1) is an anti-inflammatory and proresolving lipid mediator biosynthesized from the omega-3 fatty acid docosahexaenoic acid (DHA). Exogenous PD1 conferred protection against eosinophilic inflammation in animals with experimental asthma, although its endogenous cellular source and functions in human airways are of interest. OBJECTIVE: We sought to investigate the synthesizing capacity of PD1 in eosinophils from healthy subjects and patients with severe asthma and its direct effects on eosinophil functions. METHODS: Human eosinophil-derived metabolites of arachidonic acid and DHA were analyzed with liquid chromatography-tandem mass spectrometry-based lipidomic analysis. The biological activities of PD1 on the function of human eosinophils, including chemotaxis, adhesion molecule expressions, degranulation, superoxide anion generation, or survival, were examined. RESULTS: We identified PD1 as one of the main anti-inflammatory and proresolving molecules synthesized in human eosinophils. PD1, in nanomolar concentrations, suppressed the chemotaxis induced by CCL11/eotaxin-1 or 5-oxo-eicosatetraenoic acid and modulated the expression of the adhesion molecules CD11b and L-selectin, although it had no effects on the degranulation, superoxide anion generation, or survival of the eosinophils. Compared with the cells harvested from healthy subjects, we observed a prominent decrease in the biosynthesis of PD1 by eosinophils from patients with severe asthma, even in presence of DHA. CONCLUSION: These observations are a first indication that activated human eosinophils represent a major source of PD1, which can act as a self-resolving machinery in eosinophilic inflammation, whereas the production of PD1 is impaired in patients with severe asthma.
Assuntos
Asma/metabolismo , Ácidos Docosa-Hexaenoicos/biossíntese , Eosinófilos/metabolismo , Adulto , Idoso , Anti-Inflamatórios/metabolismo , Ácidos Araquidônicos/metabolismo , Asma/imunologia , Antígeno CD11b/metabolismo , Estudos de Casos e Controles , Moléculas de Adesão Celular/metabolismo , Quimiocina CCL11/metabolismo , Quimiotaxia/fisiologia , Ácidos Docosa-Hexaenoicos/imunologia , Eosinófilos/imunologia , Feminino , Humanos , Inflamação/metabolismo , Selectina L/metabolismo , Masculino , Neutrófilos/metabolismo , Superóxidos/metabolismoRESUMO
Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin ß9 have modes of action similar to RC21v3.
Assuntos
Candida albicans/enzimologia , Inibidores Enzimáticos/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Supressão GenéticaRESUMO
Colonization of surfaces in the human body by microorganisms is an early, essential, step in the initiation of infectious disease. We have developed in vitro assays to investigate interactions between yeast or bacterial cells and human tissues, fluids, or prostheses. Such assays can be used to identify the adhesins, ligands, and receptors involved in these interactions, for example, by determining which components of the microbe or human tissue/fluid interfere with adherence in the assay. The assays can also be applied to find ways of preventing adhesion, and subsequent disease, by investigating the effects of different conditions and added compounds on adherence in the in vitro assays. Here we describe assays for measuring adhesion of the oral yeast Candida albicans, a common commensal and opportunistic pathogen, or the bacterium Staphylococcus epidermidis, which is not normally pathogenic but is known to form biofilms on medical prostheses. The assays described belong to two approaches to investigating adhesion and biofilm formation: (i) retention at a fixed time point following liquid washes, and (ii) retention against a continuous flow of medium.
Assuntos
Candida albicans , Leveduras , Humanos , Biofilmes , Staphylococcus epidermidis , Adesinas BacterianasRESUMO
We report a case of retroperitoneal hematoma during prophylactic heparin therapy for coronavirus disease 2019 (COVID-19). A 79-year-old man was diagnosed with COVID-19 pneumonia with possible exacerbation of fibrotic hypersensitivity pneumonia. He received a prophylactic dose of subcutaneous heparin therapy, methylprednisolone pulse therapy and Intravenous remdesivir but developed a spontaneous iliopsoas muscle hematoma, and transcatheter arterial embolization was performed. Even with a prophylactic dose of subcutaneous heparin therapy, the course should be carefully monitored, especially in patients with preexisting risk factors for hemorrhagic complications. Once retroperitoneal hematoma develops, aggressive procedures, such as transcatheter arterial embolization, should be considered to avoid fatal outcomes.
Assuntos
COVID-19 , Masculino , Humanos , Idoso , Anticoagulantes/efeitos adversos , Heparina/efeitos adversos , Hematoma/induzido quimicamente , Hematoma/tratamento farmacológico , Hemorragia GastrointestinalRESUMO
Nodular pulmonary amyloidosis, a subtype of pulmonary amyloidosis, is a unique disease that can mimic lung cancer on radiographic imaging and is related to lymphoproliferative disorders. In this report, we describe a case of a 76-year-old male who presented with a solitary nodule in his left lower lung lobe on computed tomography that increased from 6 mm to 13 mm in diameter over 40 months. Lung cancer was suspected; however, transbronchial lung biopsy revealed deposition of an eosinophilic and homogeneous amorphous substance, which showed apple-green birefringence under polarized light after Congo red staining, and immunohistochemistry analysis returned positive results for immunoglobulin lambda light-chain. Upper gastrointestinal endoscopy revealed a gastric mucosa-associated lymphoid tissue (MALT) lymphoma. These findings indicated that this was a case of nodular pulmonary amyloidosis that preceded a diagnosis of MALT lymphoma.
Assuntos
Amiloidose , Neoplasias Pulmonares , Linfoma de Zona Marginal Tipo Células B , Masculino , Humanos , Idoso , Linfoma de Zona Marginal Tipo Células B/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Amiloidose/diagnóstico , Amiloidose/etiologia , Pulmão/diagnóstico por imagemRESUMO
Transforming growth factor (TGF) ß1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFß1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFß1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFß1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110ß inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL-6, calponin or smooth muscle α-actin (α-SMA) expression. However, TGFß1 increased calponin and α-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFß1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFß1 induced production of the contractile proteins calponin and α-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling.
Assuntos
Asma/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/metabolismo , Pulmão/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Asma/patologia , Benzamidas/farmacologia , Células Cultivadas , Cromonas/farmacologia , Classe I de Fosfatidilinositol 3-Quinases , Proteínas Contráteis/metabolismo , Dioxóis/farmacologia , Feminino , Humanos , Interleucina-6/genética , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Morfolinas/farmacologia , Músculo Liso/citologia , Fosfatidilinositol 3-Quinases/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Quinazolinas/farmacologia , Doadores de Tecidos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
ß(2)-Adrenergic receptor (ß2AR) agonists induce airway relaxation via cAMP. Phosphodiesterase (PDE)s degrade and regulate cAMP, and in airway smooth muscle (ASM) cells PDE4D degrades cAMP. Long-acting ß(2)-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budesonide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)-ß(1), formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with ß2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Asma/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Etanolaminas/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Budesonida/farmacologia , Estudos de Casos e Controles , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Feminino , Fumarato de Formoterol , Glucocorticoides/farmacologia , Humanos , Interleucina-6/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Receptores Adrenérgicos beta 2/metabolismo , Taquifilaxia , Adulto JovemRESUMO
OBJECTIVES: A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2. METHODS: A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined. RESULTS: Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality. CONCLUSIONS: In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded ß-1,3-glucan synthase catalytic subunits.
Assuntos
Antifúngicos/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/enzimologia , Farmacorresistência Fúngica , Equinocandinas/farmacologia , Expressão Gênica , Glucosiltransferases/biossíntese , Lipopeptídeos/farmacologia , Candida glabrata/genética , Análise Mutacional de DNA , DNA Fúngico/química , DNA Fúngico/genética , Deleção de Genes , Glucosiltransferases/genética , Micafungina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Seleção Genética , Análise de Sequência de DNARESUMO
While palliative care for patients with cancer is actively performed, it is provided only occasionally for patients with chronic non-cancerous respiratory diseases. This is due to various factors, including the fact that palliative care is not covered by health insurance and the difficulty in determining end-of-life in these patients. This paper presents two case studies to highlight the significance of palliative care team intervention for patients in the terminal stage of chronic non-cancerous respiratory diseases. Palliative care is essential to support physical problems, such as dyspnea, as well as mental disorders, such as depression, and to provide nutrition therapy and rehabilitation. To achieve care at the appropriate time in accordance with the patient's wishes, it is essential for patients to understand and accept the progress and deterioration of their disease and prepare for the end of life at an earlier stage under multidisciplinary involvement (advance care planning).
Assuntos
Neoplasias , Cuidados Paliativos , Humanos , Morte , Neoplasias/terapiaRESUMO
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are common therapeutic agents for EGFR mutation-positive advanced non-small-cell lung cancer. There has been no report of rhabdomyolysis caused by an overdose of EGFR-TKIs. We herein review the existing literature on the subject and report a rare case of rhabdomyolysis due to an overdose of gefitinib, an EGFR-TKI.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Rabdomiólise , Antineoplásicos/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/genética , Gefitinibe/efeitos adversos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/efeitos adversos , Rabdomiólise/induzido quimicamente , Rabdomiólise/tratamento farmacológicoRESUMO
Unilateral absence of the pulmonary artery (UAPA) with or without other anomalies in the heart is a rare congenital malformation. A 55-year-old Filipino woman without a remarkable medical history was admitted to our hospital for hemoptysis. Contrast-enhanced chest computed tomography revealed the absence of the left pulmonary artery. Echocardiography and right heart catheterization showed no cardiac malformations or pulmonary hypertension. We diagnosed her with isolated left-sided UAPA and performed transarterial embolization of the left inferior phrenic artery. This resolved the hemoptysis, and there was no recurrence during the four-year follow-up period.
Assuntos
Embolização Terapêutica , Cardiopatias Congênitas , Pneumopatias , Malformações Vasculares , Feminino , Hemoptise/etiologia , Hemoptise/terapia , Humanos , Pulmão/irrigação sanguínea , Pneumopatias/terapia , Pessoa de Meia-Idade , Prognóstico , Artéria Pulmonar/anormalidades , Artéria Pulmonar/diagnóstico por imagemRESUMO
Cryptogenic bilateral fibrosing pleuritis is a rare condition, and its pathogenesis and clinical course are poorly understood, with no established therapy available. A 61-year-old man presented with bilateral pleural thickening and lymphocytic exudative effusions. The patient was diagnosed with fibrosing pleuritis with no evidence of a known etiology on a surgical pleural biopsy. Within 16 months from the onset of respiratory symptoms, restrictive ventilatory impairment progressed rapidly, resulting in hypercapnic respiratory failure requiring home oxygen and non-invasive positive pressure ventilation therapies.
Assuntos
Derrame Pleural , Pleurisia , Insuficiência Respiratória , Biópsia/efeitos adversos , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Pleura/patologia , Derrame Pleural/etiologia , Pleurisia/complicações , Pleurisia/diagnóstico , Insuficiência Respiratória/complicações , Insuficiência Respiratória/terapiaRESUMO
OBJECTIVES: To analyse the clinical characteristics and prognosis of acute exacerbation (AE) in patients with idiopathic pulmonary fibrosis (IPF) and pulmonary emphysema. DESIGN: A multicentre retrospective cohort study SETTING: Two university hospitals in Japan PARTICIPANTS: Patients admitted to hospitals due to AE of IPF diagnosed based on a multidisciplinary discussion. INTERVENTIONS: None PRIMARY AND SECONDARY OUTCOME MEASURES: 90-day mortality rate METHODS: We retrospectively analysed consecutive patients with AE of IPF, with or without pulmonary emphysema, admitted to two university hospitals between 2007 and 2018. RESULTS: Among 62 patients (median age, 75 years; 48 men) admitted for AE of IPF, 29 patients (46%) presented with concomitant pulmonary emphysema. There was no significant difference in the arterial partial oxygen pressure/fraction of inhaled oxygen (P/F) ratio or other laboratory and radiographic data between patients with and without emphysema. The 90-day mortality rate was significantly lower in patients with emphysema than in those with IPF alone (23% vs 52%, p=0.03). The median survival time was significantly longer in patients with emphysema than in those with IPF alone (405 vs 242 days, p=0.02). CONCLUSION: Patients with IPF and emphysema had better short-term survival after AE than those with non-emphysematous IPF.