RESUMO
There is an urgent need for new, potent antituberculosis (anti-TB) drugs with novel mechanisms of action that can be included in new regimens to shorten the treatment period for TB. After screening a library of carbostyrils, we optimized 3,4-dihydrocarbostyril derivatives and identified OPC-167832 as having potent antituberculosis activity. The MICs of the compound for Mycobacterium tuberculosis ranged from 0.00024 to 0.002 µg/ml. It had bactericidal activity against both growing and intracellular bacilli, and the frequency of spontaneous resistance for M. tuberculosis H37Rv was less than 1.91 × 10-7 It did not show antagonistic effects with other anti-TB agents in an in vitro checkerboard assay. Whole-genome and targeted sequencing of isolates resistant to OPC-167832 identified decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), an essential enzyme for cell wall biosynthesis, as the target of the compound, and further studies demonstrated inhibition of DprE1 enzymatic activity by OPC-167832. In a mouse model of chronic TB, OPC-167832 showed potent bactericidal activities starting at a dose of 0.625 mg/kg of body weight. Further, it exhibited significant combination effects in 2-drug combinations with delamanid, bedaquiline, or levofloxacin. Finally, 3- or 4-drug regimens comprised of delamanid and OPC-167832 as the core along with bedaquiline, moxifloxacin, or linezolid showed efficacy in reducing the bacterial burden and preventing relapse superior to that of the standard treatment regimen. In summary, these results suggest that OPC-167832 is a novel and potent anti-TB agent, and regimens containing OPC-167832 and new or repurposed anti-TB drugs may have the potential to shorten the duration of treatment for TB.
Assuntos
Hidroxiquinolinas , Mycobacterium tuberculosis , Quinolonas , Animais , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , CamundongosRESUMO
Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F420)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (ndh) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Nitroimidazóis/farmacologia , Oxazóis/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Pulmonar/tratamento farmacológico , Cromatografia Líquida , Farmacorresistência Bacteriana Múltipla/genética , Isoniazida/farmacologia , Espectrometria de Massas , Ácidos Micólicos/metabolismo , NAD/análise , NADH Desidrogenase/genética , Oxirredução , Polimorfismo de Nucleotídeo Único/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
Tuberculosis (TB) treatment is long and requires multiple drugs, likely due to various phenotypes of TB bacilli with variable drug susceptibilities. Drugs with broad activity are urgently needed. This study aimed to evaluate delamanid's activity against growing or dormant bacilli in vitro as well as in vivo Cultures of Mycobacterium bovis BCG Tokyo under aerobic and anaerobic conditions were used to study the activity of delamanid against growing and dormant bacilli, respectively. Delamanid exhibited significant bactericidal activity against replicating and dormant bacilli at or above concentrations of 0.016 and 0.4 mg/liter, respectively. To evaluate delamanid's antituberculosis activity in vivo, we used a guinea pig model of chronic TB infection in which the lung lesions were similar to those in human TB disease. In the guinea pig TB model, a daily dose of 100 mg delamanid/kg of body weight for 4 or 8 weeks demonstrated strong bactericidal activity against Mycobacterium tuberculosis Importantly, histological examination revealed that delamanid killed TB bacilli within hypoxic lesions of the lung. The combination regimens containing delamanid with rifampin and pyrazinamide or delamanid with levofloxacin, ethionamide, pyrazinamide, and amikacin were more effective than the standard regimen (rifampin, isoniazid, and pyrazinamide). Our data show that delamanid is effective in killing both growing and dormant bacilli in vitro and in the guinea pig TB model. Adding delamanid to current TB regimens may improve treatment outcomes, as demonstrated in recent clinical trials with pulmonary multidrug-resistant (MDR) TB patients. Delamanid may be an important drug for consideration in the construction of new regimens to shorten TB treatment duration.
Assuntos
Antituberculosos/uso terapêutico , Nitroimidazóis/uso terapêutico , Oxazóis/uso terapêutico , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/farmacologia , Quimioterapia Combinada , Etionamida/farmacologia , Etionamida/uso terapêutico , Cobaias , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Levofloxacino/farmacologia , Levofloxacino/uso terapêutico , Pulmão/microbiologia , Pulmão/patologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Nitroimidazóis/farmacologia , Oxazóis/farmacologia , Pirazinamida/farmacologia , Pirazinamida/uso terapêutico , Rifampina/farmacologia , Rifampina/uso terapêutico , Tuberculose/microbiologiaRESUMO
Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Metiltransferases/metabolismo , RNA de Transferência/metabolismo , Proliferação de Células , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Resposta ao Choque Térmico/genética , Humanos , Metiltransferases/genética , Paclitaxel/farmacologia , Fosforilação , Estabilidade de RNA/efeitos dos fármacos , RNA de Transferência/genética , Ensaio Tumoral de Célula-TroncoRESUMO
The mitotic Aurora B kinase is overexpressed in tumors and various inhibitors for Aurora B are currently under clinical assessments. However, when considering Aurora B kinase inhibitors as anticancer drugs, their mode of action and the role of p53 status as a possible predictive factor for response still needs to be investigated. In this study, we analyzed the effects of selective Aurora B inhibition using AZD1152-HQPA/Barasertib (AZD1152) on HCT116 cells, U87-MG, corresponding isogenic p53-deficient cells and a primary glioblastoma cell line. AZD1152 treatment caused polyploidy and non-apoptotic cell death in all cell lines irrespective of p53 status and was accompanied by poly-merotelic kinetochore-microtubule attachments and DNA damage. In p53 wild-type cells a DNA damage response induced an inefficient pseudo-G1 cell cycle arrest, which was not able to halt ongoing endoreplication of cells. Of note, release of tumor cells from AZD1152 resulted in recovery of aneuploid progenies bearing numerical and structural chromosomal aberrations. Yet, AZD1152 treatment enhanced death receptor TRAIL-R2 levels in all tumor cell lines investigated. A concomitant increase of the activating natural killer (NK) cell ligand MIC A/B in p53-deficient cells and an induction of FAS/CD95 in cells containing p53 rendered AZD1152-treated cells more susceptible for NK-cell-mediated lysis. Our study mechanistically explains a p53-independent mode of action of a chemical Aurora B inhibitor and suggests a potential triggering of antitumoral immune responses, following polyploidization of tumor cells, which might constrain recovery of aneuploid tumor cells.
Assuntos
Apoptose/efeitos dos fármacos , Aurora Quinase B/biossíntese , Proliferação de Células/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Aneuploidia , Aurora Quinase B/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Organofosfatos/administração & dosagem , Quinazolinas/administração & dosagem , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteína Supressora de Tumor p53/biossínteseRESUMO
The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase-3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase-3. RhoGDIß, known as a direct cleavage substrate of caspase-3, is overexpressed in many epithelial cancers. The N-terminal-truncated RhoGDIß (ΔN-RhoGDIß) is accumulated in caspase-3-activated cells. Stable expression of ΔN-RhoGDIß in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound-healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN-RhoGDIß but not wild-type RhoGDIß was present in the detergent-soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN-RhoGDIß, resulting in increased radiation-induced compensatory proliferation linking to RhoA activation. Thus, ΔN-RhoGDIß dominant-negatively regulates Cdc42 activity and contributes to loss of polarity-related functions. The caspase-3-cleaved RhoGDIß is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493-2505, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Movimento Celular/efeitos da radiação , Polaridade Celular/efeitos da radiação , Neoplasias/patologia , Radiação , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Apoptose/efeitos da radiação , Caspase 3/metabolismo , Divisão Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Genes Dominantes , Células HeLa , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Metástase Neoplásica , Transporte Proteico/efeitos da radiação , Frações Subcelulares/metabolismo , Raios X , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The increasing global burden of multidrug-resistant tuberculosis (MDR-TB) requires reliable drug susceptibility testing that accurately characterizes susceptibility and resistance of pathogenic bacteria to effectively treat patients with this deadly disease. Delamanid is an anti-TB agent first approved in the European Union in 2014 for the treatment of pulmonary MDR-TB in adults. Using the agar proportion method, delamanid MIC was determined for 460 isolates: 316 from patients enrolled in a phase 2 global clinical trial, 76 from two phase 2 early bactericidal activity trials conducted in South Africa, and 68 isolates obtained outside clinical trials (45 from Japanese patients and 23 from South African patients). With the exception of two isolates, MICs ranged from 0.001 to 0.05 µg/ml, resulting in an MIC50 of 0.004 µg/ml and an MIC90 of 0.012 µg/ml. Various degrees of resistance to other anti-TB drugs did not affect the distribution of MICs, nor did origin of isolates from regions/countries other than South Africa. A critical concentration/breakpoint of 0.2 µg/ml can be used to define susceptible and resistant isolates based on the distribution of MICs and available pharmacokinetic data. Thus, clinical isolates from delamanid-naive patients with tuberculosis have a very low MIC for delamanid and baseline resistance is rare, demonstrating the potential potency of delamanid and supporting its use in an optimized background treatment regimen for MDR-TB.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Nitroimidazóis/farmacologia , Oxazóis/farmacologia , Farmacorresistência Bacteriana Múltipla , Humanos , Testes de Sensibilidade Microbiana , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
Superimposed epitopes, in which a shorter epitope is embedded within a longer one, can be presented by the same HLA class I molecule. CD8(+) CTL responses against such epitopes and the contribution of this phenomenon to immune control are poorly characterized. In this study, we examined HLA-A*24:02-restricted CTLs specific for the superimposed HIV Nef epitopes RYPLTFGWCF (RF10) and RYPLTFGW (RW8). Unexpectedly, RF10-specific and RW8-specific CTLs from HIV-1-infected HLA-A*24:02+ individuals had no overlapping Ag reactivity or clonotypic compositions. Single-cell TCR sequence analyses demonstrated that RF10-specific T cells had a more diverse TCR repertoire than did RW8-specific T cells. Furthermore, RF10-specific CTLs presented a higher Ag sensitivity and HIV suppressive capacity compared with RW8-specific CTLs. Crystallographic analyses revealed important structural differences between RF10- and RW8-HLA-A*24:02 complexes as well, with featured and featureless conformations, respectively, providing an explanation for the induction of distinct T cell responses against these epitopes. The present study shows that a single viral sequence containing superimposed epitopes restricted by the same HLA molecule could elicit distinct CD8+ T cell responses, therefore enhancing the control of HIV replication. This study also showed that a featured epitope (e.g., RF10) could drive the induction of T cells with high TCR diversity and affinity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Antígeno HLA-A24/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Cristalografia por Raios X , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Infecções por HIV/genética , HIV-1/genética , Antígeno HLA-A24/química , Antígeno HLA-A24/genética , Humanos , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.
Assuntos
HIV-1/imunologia , Antígenos HLA-B/imunologia , Leucócitos/imunologia , Alelos , Linfócitos T CD8-Positivos/imunologia , Estudos de Coortes , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HIV/química , Antígenos HIV/genética , Antígenos HIV/imunologia , HIV-1/genética , HIV-1/fisiologia , Antígenos HLA-B/genética , Humanos , Internacionalidade , Polimorfismo Genético , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101(+) hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101(+) hemophiliacs showed that the frequency of Pol283-8-specific CD8(+) T cells was inversely correlated with the viral load, whereas the frequencies of CD8(+) T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101(+) hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101(+) hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8(+) T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Hemofilia A/complicações , Produtos do Gene pol do Vírus da Imunodeficiência Humana/imunologia , Alelos , Povo Asiático/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Progressão da Doença , Epitopos , Infecções por HIV/complicações , Infecções por HIV/fisiopatologia , Sobreviventes de Longo Prazo ao HIV , HIV-1/fisiologia , Hemofilia A/imunologia , Hemofilia A/virologia , Humanos , Estimativa de Kaplan-Meier , Modelos Moleculares , Mutação , Peptídeos/genética , Peptídeos/imunologia , Carga Viral , Replicação ViralRESUMO
HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.
Assuntos
HIV-1/genética , HIV-1/imunologia , Evasão da Resposta Imune/genética , Mutação , Seleção Genética/imunologia , Linfócitos T Citotóxicos/imunologia , HIV-1/fisiologia , Antígenos HLA-A , Antígeno HLA-A11 , Epitopos Imunodominantes , Mutação/imunologia , Replicação ViralRESUMO
A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4+ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4+ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4+ T cells. The CD4+ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4+ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4+ T cells from an HLA-DRB1*0803+ donor. In addition, these Nef-specific cytotoxic CD4+ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4+ T cells in vitro. Nef187-203-specific cytotoxic CD4+ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803+ donors, respectively. These results suggest that HIV-1-specific CD4+ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Regulação para Baixo , Infecções por HIV/imunologia , HIV-1/fisiologia , Macrófagos/virologia , Linfócitos T Citotóxicos/imunologia , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Macrófagos/imunologia , Linfócitos T Citotóxicos/virologia , Replicação Viral/efeitos dos fármacos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/uso terapêuticoRESUMO
A 69-year-old man had experienced right chest pain for several months. Chest computed tomography (CT) showed a right upper lobe lung tumor and swelling of multiple mediastinal and right hilar lymph node. Three punctures to 4R lymph nodes and two punctures to 11i lymph nodes were performed, using endobronchial ultrasonography. Thirty days after punctures, he was admitted with appetite loss and general fatigue. Chest CT supposed the evidence of mediastinitis and pericarditis. Despite the antibiotics, cardiac tamponade developed on the third hospital day. Pericardial fenestration and pericardial drainage were performed. Gram-positive cocci were identified and Gemella sanguinis was eventually identified as the microbial identification system. Like the former reports, the necessity of surgical procedure for late onset of mediastinitis and pericarditis. caused by EBUS-TBNA was suggested.
RESUMO
There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402(+) hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Mutação , Linfócitos T Citotóxicos/imunologia , Substituição de Aminoácidos , Epitopos/genética , Epitopos/imunologia , Infecções por HIV/transmissão , HIV-1/genética , Antígenos HLA-A/imunologia , Antígeno HLA-A24 , Humanos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologiaRESUMO
The bond durability of composite-type resin cement was evaluated by means of cyclic impact tests using three different loads. In terms of experimental setup, a casting alloy, 12% Au-Pd-Ag, was used as the adherend and bonded to a cast block using a composite-type cement (Bistite II). A shear load--using plungers of three different weights at 100, 110, and 120 g--was dropped from a 3-mm height onto a small piece of the casting alloy until debonding. The cycle numbers that caused debonding were 1756 +/- 680 x 10(4) times for 100 g, 1403 +/- 515 x 10(4) times for 110 g, and 420 +/- 200 x 10(4) times for 120 g, respectively. Therefore, the group loaded with 120 g showed a significantly lower value as compared to the other two groups. On the fracture mode of the cement, it was a bulk fracture regardless of the loading weight employed in this study--the same result obtained in a previous study where heavier weights were employed.
Assuntos
Resinas Compostas/química , Colagem Dentária , Análise do Estresse Dentário/métodos , Cimentos de Resina/química , Revestimento para Fundição Odontológica/química , Corrosão Dentária , Análise do Estresse Dentário/instrumentação , Ligas de Ouro/química , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Paládio/química , Prata/química , Estresse Mecânico , Propriedades de Superfície , Temperatura , Fatores de Tempo , Suporte de CargaRESUMO
Although a limited duration of immune activation of structured treatment interruptions (STIs) has been reported, the immune escape mechanism during STIs remains obscure. We therefore investigated the role of three immunodominant cytotoxic T lymphocyte (epitopes) in 12 HLA-A*2402-positive patients participating longitudinally during the clinical study of early antiretroviral treatment (ART) with five series of structured treatment interruptions (STIs). The frequency of HLA-A*2402-restricted CTLs varied widely and a sustained CTL response was rarely noted. However, a Y-to-F substitution at the second position in an immunodominant CTL epitope Nef138-10 (Nef138-2F), which was previously demonstrated as escape mutation, was frequently detected in seven patients primarily and emerged in the remaining five patients thereafter, and the existence of escape mutations was correlated with high pVL levels early in the clinical course. These findings suggest that escape mutation in the immunodominant CTL epitope may be one of the mechanisms to limit HIV-1-specific immune control in STIs.
Assuntos
Antirretrovirais/farmacologia , Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos HLA-A/imunologia , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/imunologia , Antirretrovirais/uso terapêutico , Epitopos/genética , Epitopos/imunologia , Infecções por HIV/sangue , Infecções por HIV/virologia , Antígeno HLA-A24 , Humanos , Mutação , Estudos ProspectivosRESUMO
Delamanid, a bicyclic nitroimidazooxazole, is effective against M. tuberculosis. Previous studies have shown that resistance to a bicyclic nitroimidazooxazine, PA-824, is caused by mutations in an F420-dependent bio-activation pathway. We investigated whether the same mechanisms are responsible for resistance to delamanid. Spontaneous resistance frequencies were determined using M. bovis BCG Tokyo (BCG) and M. tuberculosis H37Rv. F420 high-performance liquid chromatography (HPLC) elution patterns of homogenates of delamanid-resistant BCG colonies and two previously identified delamanid-resistant M. tuberculosis clinical isolates were examined, followed by sequencing of genes in the F420-dependent bio-activation pathway. Spontaneous resistance frequencies to delamanid were similar to those of isoniazid and PA-824. Four distinct F420 HPLC elution patterns were observed, corresponding to colonies with mutations on fgd1, fbiA, fbiB, and fbiC with no change in the ddn mutants from the wildtype. Complementation with the wildtype sequence of the mutated gene restored susceptibility. The two delamanid-resistant clinical isolates had ddn mutations and the wildtype F420 HPLC elution pattern. In conclusion, delamanid-resistant bacilli have mutations in one of the 5 genes in the F420-dependent bio-activation pathway with distinct F420 HPLC elution patterns. Both genetic and phenotypic changes may be considered in the development of a rapid susceptibility test for delamanid.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Mutação , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Nitroimidazóis/farmacologia , Oxazóis/farmacologia , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Genótipo , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Nitrorredutases/genética , Nitrorredutases/metabolismo , Fenótipo , Riboflavina/análogos & derivados , Riboflavina/biossínteseRESUMO
Rho GDP-dissociation inhibitor ß (RhoGDIß), a regulator of the Rho family of proteins, is expressed abundantly in the hematopoietic cell lineage. During apoptosis of hematopoietic cells, RhoGDIß is cleaved by caspase3 at Asp19 and this cleaved form (Δ19RhoGDIß) has been implicated in the apoptotic pathway. To clarify the role of RhoGDIß in hematopoietic cells, the present study performed immunoblotting and immunofluorescence staining to examine the expression of RhoGDIß and ∆19RhoGDIß during phorbol 12myristate 13acetate (PMA)stimulated differentiation of human THP1 monocytic cells to macrophages. During differentiation of the THP1 cells to macrophages, the expression of RhoGDIß remained stable; however, the expression of Δ19RhoGDIß increased, particularly in wellspreading, nonapoptotic cells, which differentiated into macrophages. These results suggested that Δ19RhoGDIß has an apoptosisindependent role in the PMAinduced differentiation of THP1 cells to macrophages.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Aspártico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Linhagem Celular , Humanos , Macrófagos/efeitos dos fármacosRESUMO
BACKGROUND: Outbreaks of bloodstream infections (BSI) of nonfermenting bacteria are a critical issue and often associated with hospital environments. We experienced a long-lasting outbreak of Pseudomonas fluorescens BSI limited to a coronary care unit (CCU). METHODS: We conducted a retrospective epidemiologic investigation and a case-control study for Pseudomonas fluorescens BSI from April 2011-July 2014. Environmental sample culture was conducted to detect the specific environmental source of transmission. RESULTS: Hospital-wide microbiology data from the term identified 13 case patients with P fluorescens BSI and 32 control patients with BSI due to organisms other than P fluorescens in the CCU. The case-control study revealed that the case group had significantly higher odds of exposure to only cardiac output (CO) measurement with thermodilution method (odds ratio, 22.0; 95% confidence interval, 2.4-202.3). The organism was identified only from an ice bath used for CO measurement. The susceptibility patterns were identical among all strains derived from the cases and the environment. CONCLUSIONS: The nosocomial outbreak of P fluorescens BSI in our CCU over 2 years was associated with a contaminated ice bath used for CO measurement within the unit. Detection and elimination of the specific source was essential to stop the outbreak.
Assuntos
Bacteriemia/microbiologia , Unidades de Cuidados Coronarianos , Infecção Hospitalar/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas fluorescens , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/epidemiologia , Estudos de Casos e Controles , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Contaminação de Equipamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/epidemiologia , Estudos Retrospectivos , Adulto JovemRESUMO
The mechanistic basis for the progressive accumulation of Y(135)F Nef mutant viruses in the HIV-1-infected population remains poorly understood. Y(135)F viruses carry the 2F mutation within RW8 and RF10, which are two HLA-A(∗)24:02-restricted superimposed Nef epitopes recognized by distinct and adaptable CD8(+) T cell responses. We combined comprehensive analysis of the T cell receptor repertoire and cross-reactive potential of wild-type or 2F RW8- and RF10-specific CD8(+) T cells with peptide-MHC complex stability and crystal structure studies. We find that, by affecting direct and water-mediated hydrogen bond networks within the peptide-MHC complex, the 2F mutation reduces both TCR and HLA binding. This suggests an advantage underlying the evolution of the 2F variant with decreased CD8(+) T cell efficacy. Our study provides a refined understanding of HIV-1 and CD8(+) T cell co-adaptation at the population level.