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1.
Vaccines (Basel) ; 9(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34451973

RESUMO

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

2.
Vaccines (Basel) ; 9(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916180

RESUMO

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.

3.
Sci Rep ; 10(1): 16257, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004826

RESUMO

Tuberculosis (TB) is a chronic inflammatory disease that is often associated with alterations in systemic and cellular metabolism that resolves following successful antimicrobial drug treatment. We hypothesized that altered systemic glucose metabolism as a consequence of Mycobacterium tuberculosis (Mtb) infection, contributes to TB pathogenesis, and when normalized with anti-glycemic drugs would improve clinical outcomes. To test this hypothesis, guinea pigs were treated daily with the anti-diabetic drug metformin starting 4 weeks prior or concurrent with aerosol exposure to the H37Rv strain of Mtb. In the chronic stages of infection, Mtb infected metformin-treated animals had restored systemic insulin sensitivity but remained glucose intolerant as determined by oral glucose tolerance testing. Despite persistent glucose intolerance, metformin-treated guinea pigs had a 2.8-fold reduction in lung lesion burden and a 0.7 log decrease in CFUs. An alternative hypothesis that metformin treatment improved clinical disease by having a direct effect on immune cell energy metabolism was tested using extracellular flux analysis and flow cytometry. The proinflammatory immune response to Mtb infection in untreated guinea pigs was associated with a marked increase in energy metabolism (glycolysis and mitochondrial respiration) of peripheral blood mononuclear cells (PBMCs), which was normalized in metformin-treated guinea pigs. Moreover, both CD4+ and CD8+ T lymphocytes from Mtb infected, metformin treated animals maintained a more normal mitochondrial membrane potential while those isolated from untreated animals had persistent mitochondrial hyperpolarization. These data suggest that metformin promotes natural host resistance to Mtb infection by maintaining immune cell metabolic homeostasis and function during the chronic stages of active TB disease.


Assuntos
Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Animais , Citocinas/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Intolerância à Glucose/tratamento farmacológico , Cobaias , Resistência à Insulina , Pulmão/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Superóxidos/metabolismo , Linfócitos T/metabolismo , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologia
4.
Vaccine ; 38(45): 7156-7165, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32978002

RESUMO

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.


Assuntos
Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose , Sistemas de Secreção Tipo VII , Animais , Antígenos de Bactérias , Vacina BCG , Cobaias , Imunização Secundária , Camundongos , Mycobacterium tuberculosis/genética , Tuberculose/prevenção & controle , Vacinação
5.
Sci Rep ; 10(1): 7651, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32377001

RESUMO

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline's modularity allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confirmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing flow cytometry regardless of cytometer or panel design. The code is available at https://github.com/aef1004/cyto-feature_engineering.


Assuntos
Citodiagnóstico/métodos , Suscetibilidade a Doenças/imunologia , Citometria de Fluxo , Animais , Biomarcadores , Células Sanguíneas/metabolismo , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Camundongos , Mycobacterium tuberculosis/imunologia , Fenótipo , Tuberculose/diagnóstico , Tuberculose/imunologia , Tuberculose/microbiologia
6.
Curr Protoc Cytom ; 93(1): e74, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32421215

RESUMO

Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.


Assuntos
Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Animais , Contagem de Células , Espaço Intracelular/metabolismo , Pulmão/citologia , Camundongos Endogâmicos C57BL , Análise de Célula Única , Baço/citologia , Linfócitos T/imunologia
7.
Vet Immunol Immunopathol ; 215: 109884, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420066

RESUMO

Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48 h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.


Assuntos
Vacina BCG/imunologia , Modelos Animais de Doenças , Mycobacterium tuberculosis/imunologia , Porco Miniatura/imunologia , Tuberculose Pulmonar/imunologia , Animais , Animais Recém-Nascidos , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Imunogenicidade da Vacina , Memória Imunológica , Imunofenotipagem , Estudos Longitudinais , Ativação Linfocitária , Masculino , Monócitos/imunologia , Suínos , Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle
8.
Sci Rep ; 9(1): 1513, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30728417

RESUMO

There is an urgent need to develop new drugs against tuberculosis. In particular, it is critical to target drug tolerant Mycobacterium tuberculosis (M. tuberculosis), responsible, in part, for the lengthy antibiotic regimen required for treatment. We previously postulated that the presence of in vivo biofilm-like communities of M. tuberculosis could contribute to this drug tolerance. Consistent with this hypothesis, certain 2-aminoimidazole (2-AIs) molecules with anti-biofilm activity were shown to revert mycobacterial drug tolerance in an in vitro M. tuberculosis biofilm model. While exploring their mechanism of action, it was serendipitously observed that these 2-AI molecules also potentiated ß-lactam antibiotics by affecting mycobacterial protein secretion and lipid export. As these two bacterial processes are energy-dependent, herein it was evaluated if 2-AI compounds affect mycobacterial bioenergetics. At low concentrations, 2B8, the lead 2-AI compound, collapsed both components of the proton motive force, similar to other cationic amphiphiles. Interestingly, however, the minimum inhibitory concentration of 2B8 against M. tuberculosis correlated with a higher drug concentration determined to interfere with the mycobacterial electron transport chain. Collectively, this study elucidates the mechanism of action of 2-AIs against M. tuberculosis, providing a tool to better understand mycobacterial bioenergetics and develop compounds with improved anti-mycobacterial activity.


Assuntos
Biofilmes/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Imidazóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Força Próton-Motriz/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Antituberculosos/farmacologia , Biofilmes/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Consumo de Oxigênio/efeitos dos fármacos , Tuberculose/microbiologia
9.
Tuberculosis (Edinb) ; 106: 91-98, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28802411

RESUMO

In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Current animal models study TB during adulthood but animal models for infant TB are scarce. Here we propose that minipigs can be used as an animal model to study adult, adolescent and infant TB including natural transmission. In these studies, two-month old minipigs (representing infant age in humans) and six-month old minipigs (representing adolescence in humans) were infected via the aerosol route with hyper-virulent clinical strain W-Beijing Mycobacterium tuberculosis (Mtb) HN878 and were monitored for 11 or 36 weeks post-challenge, respectively. In the same studies, infected and unchallenged animals were housed together. Viable bacteria were recovered from pulmonary and thoracic lymph nodes from both -infected and their initially unchallenged natural contacts. Bacillary load, gross lesions and histopathology revealed similarities to the spectrum of disease observed in human TB. The study did not reach terminal end point, thus it was not possible to annotate definitive clinical symptoms of active TB. The results demonstrated that minipigs are experimental hosts of Mtb HN878, and the pathology developed in their lungs resembles pathological findings described in human TB. Importantly, within communities of Mtb infected minipigs natural transmission occurs.


Assuntos
Mycobacterium tuberculosis/patogenicidade , Tuberculose dos Linfonodos/microbiologia , Tuberculose dos Linfonodos/transmissão , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/transmissão , Aerossóis , Fatores Etários , Animais , Carga Bacteriana , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Exposição por Inalação , Pulmão/microbiologia , Pulmão/patologia , Linfonodos/microbiologia , Linfonodos/patologia , Suínos , Porco Miniatura , Fatores de Tempo , Tuberculose dos Linfonodos/patologia , Tuberculose Pulmonar/patologia
10.
PLoS One ; 12(7): e0180925, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28749949

RESUMO

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment.


Assuntos
Anti-Infecciosos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Imidazóis/farmacologia , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/enzimologia , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia , Carbenicilina/farmacologia , Corantes/química , Lipídeos/análise , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ácidos Nucleicos/metabolismo , Penicilina V/farmacologia , Dodecilsulfato de Sódio/farmacologia , Coloração e Rotulagem , Transcrição Genética/efeitos dos fármacos , Vancomicina/farmacologia
11.
Environ Microbiol ; 19(10): 4010-4021, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28585299

RESUMO

Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis. Despite intensive TB control campaigns, there are sporadic outbreaks of bovine TB in regions declared TB free. It is unclear how M. bovis is able to survive in the environment for long periods of time. We hypothesized that Free-living amoebae (FLA), as ubiquitous inhabitants of soil and water, may act as long-term reservoirs of M. bovis in the environment. In our model, M. bovis would be taken up by amoebal trophozoites, which are the actively feeding, replicating and mobile form of FLA. Upon exposure to hostile environmental conditions, infected FLA will encyst and provide an intracellular niche allowing their M. bovis cargo to persist for extended periods of time. Here, we show that five FLA species (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba lenticulata, Vermamoeba vermiformis and Dictyostellium discoideum) are permissive to M. bovis infection and that the M. bovis bacilli may survive within the cysts of four of these species for over 60 days. We further show that exposure of M. bovis-infected trophozoites and cysts to Balb/c mice leads to pulmonary TB. This work describes for the first time that FLA carrying M. bovis can transmit TB.


Assuntos
Amebozoários/microbiologia , Reservatórios de Doenças/microbiologia , Mycobacterium bovis/crescimento & desenvolvimento , Acanthamoeba/microbiologia , Animais , Bovinos , Dictyostelium/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/patogenicidade , Tuberculose Bovina/microbiologia , Tuberculose Bovina/transmissão
12.
Angew Chem Int Ed Engl ; 56(14): 3940-3944, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28247991

RESUMO

A library of 2-aminobenzimidazole derivatives was screened for the ability to suppress ß-lactam resistance in Mycobacterium smegmatis. Several non-bactericidal compounds were identified that reversed intrinsic resistance to ß-lactam antibiotics in a manner distinct from ß-lactamase inhibitors. Activity also translates to M. tuberculosis, with a lead compound from this study potently suppressing carbenicillin resistance in multiple M. tuberculosis strains (including multidrug-resistant strains). Preliminary mechanistic studies revealed that the lead compounds act through a mechanism distinct from that of traditional ß-lactamase inhibitors.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Lactamas/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Benzimidazóis/química , Descoberta de Drogas , Lactamas/síntese química , Lactamas/química , Estrutura Molecular , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismo
13.
Antimicrob Agents Chemother ; 60(9): 5198-207, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27297488

RESUMO

In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Pulmão/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Animais , Carga Bacteriana/efeitos dos fármacos , Transporte Biológico , Ciprofloxacina/farmacologia , Modelos Animais de Doenças , Doxiciclina/farmacologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Isoniazida/farmacologia , Pulmão/microbiologia , Pulmão/patologia , Proteínas de Membrana Transportadoras/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/patologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
14.
J Leukoc Biol ; 99(4): 605-17, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26590147

RESUMO

Increasing information has shown that many newly emerging strains of Mycobacterium tuberculosis, including the highly prevalent and troublesome Beijing family of strains, can potently induce the emergence of Foxp3(+)CD4 Tregs Although the significance of this is still not fully understood, we have previously provided evidence that the emergence of this population can significantly ablate the protective effect of BCG vaccination, causing progressive fatal disease in the mouse model. However, whether the purpose of this response is to control inflammation or to directly dampen the acquired immune response is still unclear. In the present study, we have shown, using both cell depletion and adoptive transfer strategies, that Tregs can have either properties. Cell depletion resulted in a rapid, but transient, decrease in the lung bacterial load, suggesting release or temporary re-expansion of effector immunity. Transfer of Tregs into Rag2(-/-)or marked congenic mice worsened the disease course and depressed cellular influx of effector T cells into the lungs. Tregs from infected donors seemed to preferentially depress the inflammatory response and granulocytic influx. In contrast, those from BCG-vaccinated and then challenged donors seemed more focused on depression of acquired immunity. These qualitative differences might be related to increasing knowledge reflecting the plasticity of the Treg response.


Assuntos
Imunidade Celular/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinação , Animais , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/patologia , Tuberculose/genética
15.
Antimicrob Agents Chemother ; 59(11): 6904-12, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26303795

RESUMO

Over the last 10 years, Mycobacterium abscessus group strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections with M. abscessus are often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection with M. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an optimal model for compound screening. Thus, we set out to assess the antimycobacterial activity of clarithromycin, clofazimine, bedaquiline, and clofazimine-bedaquiline combinations against M. abscessus-infected GKO and SCID murine infection models. Treatment of GKO and SCID mice with a combination of clofazimine and bedaquiline was the most effective in decreasing the M. abscessus organ burden.


Assuntos
Antibacterianos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Mycobacterium/efeitos dos fármacos , Animais , Claritromicina/farmacologia , Clofazimina/farmacologia , Diarilquinolinas/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Camundongos Knockout , Camundongos SCID , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium
16.
J Antibiot (Tokyo) ; 68(7): 453-62, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25649856

RESUMO

The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.


Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Canais Iônicos/antagonistas & inibidores , Mecanotransdução Celular/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Caenorhabditis elegans , Linhagem Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento
17.
Clin Vaccine Immunol ; 22(1): 91-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25392011

RESUMO

The global epidemic caused by the bacterial pathogen Mycobacterium tuberculosis continues unabated. Moreover, the only available vaccine against tuberculosis, Mycobacterium bovis bacillus Calmette-Guérin (BCG), demonstrates variable efficacy. To respond to this global threat, new animal models that mimic the pathological disease process in humans are required for vaccine testing. One new model, susceptible C3Heb/FeJ mice, is similar to human tuberculosis in that these animals are capable of forming necrotic tubercle granulomas, in contrast to resistant C3H/HeOuJ mice. In this study, we evaluated the impact of prior BCG vaccination of C3Heb/FeJ and C3H/HeOuJ mice on exposure to a low-dose aerosol of Mycobacterium tuberculosis W-Beijing strain SA161. Both BCG-vaccinated murine strains demonstrated reduced bacterial loads 25 days after infection compared to controls, indicating vaccine efficacy. However, during chronic infection, vaccine efficacy waned in C3H/HeOuJ but not in C3Heb/FeJ mice. Protection in vaccinated C3Heb/FeJ mice was associated with reduced numbers of CD11b(+) Gr1(+) cells, increased numbers of effector and memory T cells, and an absence of necrotic granulomas. BCG vaccine efficacy waned in C3H/HeOuJ mice, as indicated by reduced expression of gamma interferon (IFN-γ) and increased expressions of interleukin-17 (IL-17), IL-10, and Foxp3 by T cells compared to C3Heb/FeJ mice. This is the first murine vaccine model system described to date that can be utilized to dissect differential vaccine-derived immune efficacy.


Assuntos
Vacina BCG/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/prevenção & controle , Animais , Vacina BCG/administração & dosagem , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/análise , Memória Imunológica , Imunofenotipagem , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Camundongos Endogâmicos C3H , Subpopulações de Linfócitos T , Linfócitos T/imunologia
18.
EMBO Mol Med ; 7(2): 127-39, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25535254

RESUMO

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection.


Assuntos
Anticonvulsivantes/administração & dosagem , Antituberculosos/administração & dosagem , Autofagia/efeitos dos fármacos , Carbamazepina/administração & dosagem , Inositol/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose/fisiopatologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Tuberculose/imunologia , Tuberculose/metabolismo , Peixe-Zebra
19.
Am J Pathol ; 184(4): 1104-1118, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24492198

RESUMO

Impaired glucose tolerance and type 2 diabetes were induced in guinea pigs to model the emerging comorbidity of Mycobacterium tuberculosis infection in diabetic patients. Type 2 diabetes mellitus was induced by low-dose streptozotocin in guinea pigs rendered glucose intolerant by first feeding a high-fat, high-carbohydrate diet before M. tuberculosis exposure. M. tuberculosis infection of diabetic guinea pigs resulted in severe and rapidly progressive tuberculosis (TB) with a shortened survival interval, more severe pulmonary and extrapulmonary pathology, and a higher bacterial burden compared with glucose-intolerant and nondiabetic controls. Compared with nondiabetics, diabetic guinea pigs with TB had an exacerbated proinflammatory response with more severe granulocytic inflammation and higher gene expression for the cytokines/chemokines interferon-γ, IL-17A, IL-8, and IL-10 in the lung and for interferon-γ, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1 in the spleen. TB disease progression in guinea pigs with impaired glucose tolerance was similar to that of nondiabetic controls in the early stages of infection but was more severe by day 90. The guinea pig model of type 2 diabetes-TB comorbidity mimics important features of the naturally occurring disease in humans. This model will be beneficial in understanding the complex pathogenesis of TB in diabetic patients and to test new strategies to improve TB and diabetes control when the two diseases occur together.


Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologia , Tuberculose/complicações , Tuberculose/imunologia , Animais , Comorbidade , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Citometria de Fluxo , Cobaias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tuberculose/patologia
20.
PLoS One ; 8(11): e80669, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24224058

RESUMO

BACKGROUND: Tuberculosis is one of the world's leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1(int)CD11b(+) cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design. METHODOLOGY/PRINCIPAL FINDINGS: We compared the bacterial burden, lung pathology and Gr1(int)CD11b(+) myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1(+) cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1(+)CD11b(+) cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1(hi) and Gr1(int) populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1(int) populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1(hi) and Gr1(int) cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4(+) T to Gr1(+) cells increased. Our results illustrate a yet unrecognized interplay between Gr1(+) cells and CD4(+) T cells in tuberculosis.


Assuntos
Antígeno CD11b/metabolismo , Infecções por Mycobacterium/metabolismo , Mycobacterium tuberculosis/patogenicidade , Receptores de Quimiocinas/metabolismo , Animais , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética
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