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1.
Immunology ; 163(4): 431-435, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34002402

RESUMO

The pandemic has brought challenges to teaching lab and research skills. Here Nigel Francis and colleagues explore the diverse approaches taken to replace lab-based immunology teaching, explain how networks of educators have driven this innovation and discuss the importance of retaining best practice into the future.


Assuntos
Alergia e Imunologia/educação , Pesquisa Biomédica/educação , COVID-19 , Instrução por Computador , Educação a Distância , Laboratórios , Estudantes , Ensino , Currículo , Humanos
2.
Appl Environ Microbiol ; 86(20)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32801179

RESUMO

Staphylococcus pseudintermedius causes opportunistic infections in dogs. It also has significant zoonotic potential, with the emergence of multidrug resistance leading to difficulty treating both animal and human infections. Manuka honey has previously been reported to inhibit many bacterial pathogens, including methicillin-resistant Staphylococcus aureus, and is successfully utilized in both clinical and veterinary practice. Here, we evaluated the ability of manuka honey to inhibit strains of S. pseudintermedius grown alone and in combination with antibiotics, as well as its capacity to modulate virulence within multiple S. pseudintermedius isolates. All 18 of the genetically diverse S. pseudintermedius strains sequenced and tested were inhibited by ≤12% (wt/vol) medical-grade manuka honey, although tolerance to five clinically relevant antibiotics was observed. The susceptibility of the isolates to four of these antibiotics was significantly increased (P ≤ 0.05) when combined with sublethal concentrations of honey, although sensitivity to oxacillin was decreased. Virulence factor (DNase, protease, and hemolysin) activity was also significantly reduced (P ≤ 0.05) in over half of isolates when cultured with sublethal concentrations of honey (13, 9, and 10 isolates, respectively). These findings highlight the potential for manuka honey to be utilized against S. pseudintermedius infections.IMPORTANCEStaphylococcus pseudintermedius is an important member of the skin microbial community in animals and can cause opportunistic infections in both pets and their owners. The high incidence of antimicrobial resistance in S. pseudintermedius highlights that this opportunistic zoonotic pathogen can cause infections which require prolonged and intensive treatment to resolve. Manuka honey has proven efficacy against many bacterial pathogens and is an accepted topical treatment for infections in both veterinary and clinical practice, and so it is a particularly appropriate antimicrobial for use with zoonotic pathogens such as S. pseudintermedius Here, we demonstrate that not only is manuka honey highly potent against novel multidrug-resistant S. pseudintermedius isolates, it also acts synergistically with clinically relevant antibiotics. In addition, manuka honey modulates S. pseudintermedius virulence activity, even at subinhibitory concentrations. In a clinical setting, these attributes may assist in controlling infection, allowing a more rapid resolution and reducing antibiotic use.


Assuntos
Antibacterianos/farmacologia , Mel/análise , Staphylococcus/efeitos dos fármacos , Antibacterianos/análise , Staphylococcus/genética , Staphylococcus/patogenicidade , Staphylococcus/fisiologia , Virulência/efeitos dos fármacos
3.
Part Fibre Toxicol ; 16(1): 8, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760282

RESUMO

BACKGROUND: It is well established that toxicological evaluation of engineered nanomaterials (NMs) is vital to ensure the health and safety of those exposed to them. Further, there is a distinct need for the development of advanced physiologically relevant in vitro techniques for NM hazard prediction due to the limited predictive power of current in vitro models and the unsustainability of conducting nano-safety evaluations in vivo. Thus, the purpose of this study was to develop alternative in vitro approaches to assess the potential of NMs to induce genotoxicity by secondary mechanisms. RESULTS: This was first undertaken by a conditioned media-based technique, whereby cell culture media was transferred from differentiated THP-1 (dTHP-1) macrophages treated with γ-Fe2O3 or Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) to the bronchial cell line 16HBE14o-. Secondly construction and SPION treatment of a co-culture model comprising of 16HBE14o- cells and dTHP-1 macrophages. For both of these approaches no cytotoxicity was detected and chromosomal damage was evaluated by the in vitro micronucleus assay. Genotoxicity assessment was also performed using 16HBE14o- monocultures, which demonstrated only γ-Fe2O3 nanoparticles to be capable of inducing chromosomal damage. In contrast, immune cell conditioned media and dual cell co-culture SPION treatments showed both SPION types to be genotoxic to 16HBE14o- cells due to secondary genotoxicity promoted by SPION-immune cell interaction. CONCLUSIONS: The findings of the present study demonstrate that the approach of using single in vitro cell test systems precludes the ability to consider secondary genotoxic mechanisms. Consequently, the use of multi-cell type models is preferable as they better mimic the in vivo environment and thus offer the potential to enhance understanding and detection of a wider breadth of potential damage induced by NMs.


Assuntos
Dano ao DNA , Compostos Férricos/toxicidade , Nanopartículas de Magnetita/toxicidade , Testes de Mutagenicidade/métodos , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Citocinas/biossíntese , Endocitose/efeitos dos fármacos , Humanos , Técnicas In Vitro , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Células THP-1
4.
BMC Biol ; 16(1): 84, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30071832

RESUMO

BACKGROUND: Helicobacter pylori are stomach-dwelling bacteria that are present in about 50% of the global population. Infection is asymptomatic in most cases, but it has been associated with gastritis, gastric ulcers and gastric cancer. Epidemiological evidence shows that progression to cancer depends upon the host and pathogen factors, but questions remain about why cancer phenotypes develop in a minority of infected people. Here, we use comparative genomics approaches to understand how genetic variation amongst bacterial strains influences disease progression. RESULTS: We performed a genome-wide association study (GWAS) on 173 H. pylori isolates from the European population (hpEurope) with known disease aetiology, including 49 from individuals with gastric cancer. We identified SNPs and genes that differed in frequency between isolates from patients with gastric cancer and those with gastritis. The gastric cancer phenotype was associated with the presence of babA and genes in the cag pathogenicity island, one of the major virulence determinants of H. pylori, as well as non-synonymous variations in several less well-studied genes. We devised a simple risk score based on the risk level of associated elements present, which has the potential to identify strains that are likely to cause cancer but will require refinement and validation. CONCLUSION: There are a number of challenges to applying GWAS to bacterial infections, including the difficulty of obtaining matched controls, multiple strain colonization and the possibility that causative strains may not be present when disease is detected. Our results demonstrate that bacterial factors have a sufficiently strong influence on disease progression that even a small-scale GWAS can identify them. Therefore, H. pylori GWAS can elucidate mechanistic pathways to disease and guide clinical treatment options, including for asymptomatic carriers.


Assuntos
Variação Genética , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Helicobacter pylori/genética , Neoplasias Gástricas/microbiologia , Gastrite/etiologia , Humanos , Metaplasia/etiologia , Polimorfismo de Nucleotídeo Único , Risco , Neoplasias Gástricas/epidemiologia , Fatores de Virulência/genética
5.
Int J Med Microbiol ; 307(7): 382-387, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28826573

RESUMO

Staphylococcus epidermidis is a common cause of biomedical device-associated infections. Agr is the major quorum sensing system in staphylococci and regulates virulence factors. Four agr-specificity groups exist in S. epidermidis, and chronic S. epidermidis infections are hypothesised to select for agr-negative phenotypes. Therefore, we investigated S. epidermidis strains from prosthetic joint- and catheter-associated infections to establish i) whether an infection selects for an agr-negative phenotype; ii) the importance of PSMγ and iii) if the agr-specificity group is infection dependent. S. epidermidis nasal isolates from healthy volunteers were used as controls. The distribution of agr-specificity groups was significantly different between infection and control episodes, but did not distinguish between the infection types. PSMγ secretion was used to determine agr-activity and HPLC analysis showed that 44% of prosthetic and 32% of catheter-associated episodes produced no PSMγ in comparison to 8% of the control strains. However, PSMγ expression did not always correlate with RNAIII up-regulation, indicating that PSMγ synthesis is likely influenced by additional post-transcriptional control. The data suggests chronic S. epidermidis infections favour agr-specificity group 1 but the results suggest that they do not select for an agr-negative phenotype. Further studies are required to explore the mechanisms underlying the selection and survival of these S. epidermidis phenotypes isolated from biomedical device-associated infections.


Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas a Cateter/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Biomarcadores , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Bacteriano/fisiologia , Staphylococcus epidermidis/patogenicidade
6.
Mutagenesis ; 32(1): 233-241, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27815329

RESUMO

With the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity.


Assuntos
Dano ao DNA , Testes de Mutagenicidade/métodos , Nanopartículas/toxicidade , Animais , DNA/efeitos dos fármacos , Humanos
7.
Int J Med Microbiol ; 305(8): 948-56, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26365169

RESUMO

BACKGROUND: A major complication of using medical devices is the development of biofilm-associated infection caused by Staphylococcus epidermidis where polysaccharide intercellular adhesin (PIA) is a major mechanism of biofilm accumulation. PIA affects innate and humoral immunity in isolated cells and animal models. Few studies have examined these effects in prosthetic joint infection (PJI). METHODS: This study used ex vivo whole blood modelling in controls together with matched-serum and staphylococcal isolates from patients with PJI. RESULTS: Whole blood killing of PIA positive S. epidermidis and its isogenic negative mutant was identical. Differences were unmasked in immunosuppressed whole blood pre-treated with dexamethasone where PIA positive bacteria showed a more resistant phenotype. PIA expression was identified in three unique patterns associated with bacteria and leukocytes, implicating a soluble form of PIA. Purified PIA reduced whole blood killing while increasing C5a levels. In clinically relevant staphylococcal isolates and serum samples from PJI patients; firstly complement C5a was increased 3-fold compared to controls; secondly, the C5a levels were significantly higher in serum from PJI patients whose isolates preferentially formed PIA-associated biofilms. CONCLUSIONS: These data demonstrate for the first time that the biological effects of PIA are mediated through C5a in patients with PJI.


Assuntos
Artrite/microbiologia , Atividade Bactericida do Sangue , Complemento C5a/metabolismo , Interações Hospedeiro-Patógeno , Polissacarídeos Bacterianos/metabolismo , Infecções Relacionadas à Prótese/microbiologia , Staphylococcus epidermidis/fisiologia , Humanos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/metabolismo
8.
Biomedicines ; 12(4)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38672079

RESUMO

Whole blood models are rapid and versatile for determining immune responses to inflammatory and infectious stimuli, but they have not been used for bacterial discrimination. Staphylococcus aureus, S. epidermidis and Escherichia coli are the most common causes of invasive disease, and rapid testing strategies utilising host responses remain elusive. Currently, immune responses can only discriminate between bacterial 'domains' (fungi, bacteria and viruses), and very few studies can use immune responses to discriminate bacteria at the species and strain level. Here, whole blood was used to investigate the relationship between host responses and bacterial strains. Results confirmed unique temporal profiles for the 10 parameters studied: IL-6, MIP-1α, MIP-3α, IL-10, resistin, phagocytosis, S100A8, S100A8/A9, C5a and TF3. Pairwise analysis confirmed that IL-6, resistin, phagocytosis, C5a and S100A8/A9 could be used in a discrimination scheme to identify to the strain level. Linear discriminant analysis (LDA) confirmed that (i) IL-6, MIP-3α and TF3 could predict genera with 95% accuracy; (ii) IL-6, phagocytosis, resistin and TF3 could predict species at 90% accuracy and (iii) phagocytosis, S100A8 and IL-10 predicted strain at 40% accuracy. These data are important because they confirm the proof of concept that host biomarker panels could be used to identify bacterial pathogens.

9.
Biomedicines ; 12(1)2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38255233

RESUMO

The exposure of human lung and skin to carbon black (CB) is continuous due to its widespread applications. Current toxicological testing uses 'healthy' cellular systems; however, questions remain whether this mimics the everyday stresses that human cells are exposed to, including infection. Staphylococcus aureus lung and skin infections remain prevalent in society, and include pneumonia and atopic dermatitis, respectively, but current in vitro toxicological testing does not consider infection stress. Therefore, investigating the effects of CB co-exposure in 'stressed' infected epithelial cells in vitro may better approximate true toxicity. This work aims to study the impact of CB exposure during Staphylococcus aureus infection stress in A549 (lung) and HaCaT (skin) epithelial cells. Physicochemical characterisation of CB confirmed its dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth in cell culture media. CB did not induce cytokines or antimicrobial peptides from lung and skin epithelial cells, when given alone, but did reduce HaCaT and A549 cell viability to 55% and 77%, respectively. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both lung and skin epithelial cells. IL-6 and human beta defensin (hßD)-2 could only be detected when cells were stimulated with S. aureus with no decreases in cell viability. However, co-exposure to CB (100 µg/mL) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus alone) without further reduction in cell viability. Furthermore, the same co-exposure induced significantly more hßD-2 (compared to S. aureus alone). This work confirms that toxicological testing in healthy versus stressed cells gives significantly different responses. This has significant implications for toxicological testing and suggests that cell stresses (including infection) should be included in current models to better represent the diversity of cell viabilities found in lung and skin within a general population. This model will have significant application when estimating CB exposure in at-risk groups, such as factory workers, the elderly, and the immunocompromised.

10.
Blood ; 117(19): 5178-88, 2011 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-21292772

RESUMO

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kδ. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.


Assuntos
Complemento C5a/imunologia , Estado Terminal , Infecção Hospitalar/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Actinas/imunologia , Actinas/metabolismo , Separação Celular , Infecção Hospitalar/epidemiologia , Citometria de Fluxo , Humanos , Polimerização , Proteína rhoA de Ligação ao GTP/imunologia , Proteína rhoA de Ligação ao GTP/metabolismo
11.
BMC Microbiol ; 12: 76, 2012 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-22594478

RESUMO

BACKGROUND: The skin commensal and opportunistic pathogen Staphylococcus epidermidis is a leading cause of hospital-acquired and biomaterial-associated infections. The polysaccharide intercellular adhesin (PIA), a homoglycan composed of ß-1,6-linked N-acetylglucosamine residues, synthesized by enzymes encoded in icaADBC is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated S. epidermidis infection. Extracellular mucous layer extracts of S. epidermidis contain another major polysaccharide, referred to as 20-kDa polysaccharide (20-kDaPS), composed mainly out of glucose, N-acetylglucosamine, and being partially sulfated. 20-kDaPS antiserum prevents adhesion of S. epidermidis on endothelial cells and development of experimental keratitis in rabbits. Here we provide experimental evidence that 20-kDaPS and PIA represent distinct molecules and that 20-kDaPS is implicated in endocytosis of S. epidermidis bacterial cells by human monocyte-derived macrophages. RESULTS: Analysis of 75 clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kDaPS is expressed exclusively in S. epidermidis but not in other coagulase-negative staphylococcal species. Tn917-insertion in various locations in icaADBC in mutants M10, M22, M23, and M24 of S. epidermidis 1457 are abolished for PIA synthesis, while 20-kDaPS expression appears unaltered as compared to wild-type strains using specific anti-PIA and anti-20-kDaPS antisera. While periodate oxidation and dispersin B treatments abolish immuno-reactivity and intercellular adhesive properties of PIA, no abrogative activity is exerted towards 20-kDaPS immunochemical reactivity following these treatments. PIA polysaccharide I-containing fractions eluting from Q-Sepharose were devoid of detectable 20-kDaPS using specific ELISA. Preincubation of non-20-kDaPS-producing clinical strain with increasing amounts of 20-kDaPS inhibits endocytosis by human macrophages, whereas, preincubation of 20-kDaPS-producing strain ATCC35983 with 20-kDaPS antiserum enhances bacterial endocytosis by human macrophages. CONCLUSIONS: In conclusion, icaADBC is not involved in 20-kDaPS synthesis, while the chemical and chromatographic properties of PIA and 20-kDaPS are distinct. 20-kDaPS exhibits anti-phagocytic properties, whereas, 20-kDaPS antiserum may have a beneficial effect on combating infection by 20-kDaPS-producing S. epidermidis.


Assuntos
Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose , Polissacarídeos Bacterianos/metabolismo , Staphylococcus epidermidis/imunologia , Staphylococcus epidermidis/metabolismo , Células Cultivadas , Elementos de DNA Transponíveis , Perfilação da Expressão Gênica , Humanos , Peso Molecular , Mutagênese Insercional , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/imunologia
12.
Am J Respir Crit Care Med ; 183(4): 491-9, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20870753

RESUMO

RATIONALE: Bronchiectasis is a chronic debilitating disease with few evidence-based long-term treatments. OBJECTIVES: A randomized controlled trial assessing the efficacy of nebulized gentamicin therapy over 1 year in patients with non-cystic fibrosis bronchiectasis. METHODS: Sixty-five patients were randomized to either twice-daily nebulized gentamicin, 80 mg, or nebulized 0.9% saline, for 12 months. All were reviewed at three-monthly intervals during treatment and at 3 months' follow-up. MEASUREMENTS AND MAIN RESULTS: At each review the following were assessed: quantitative and qualitative sputum bacteriology; sputum purulence and 24-hour volume; FEV(1), FVC, and forced expiratory flow, midexpiratory phase; exercise capacity; Leicester Cough Questionnaire and St. George's Respiratory Questionnaire; and exacerbation frequency. Fifty-seven patients completed the study. At the end of 12 months' treatment, compared with the saline group, in the gentamicin group there was reduced sputum bacterial density with 30.8% eradication in those infected with Pseudomonas aeruginosa and 92.8% eradication in those infected with other pathogens; less sputum purulence (8.7% vs. 38.5%; P < 0.0001); greater exercise capacity (510 [350-690] m vs. 415 [267.5-530] m; P = 0.03); and fewer exacerbations (0 [0-1] vs. 1.5 [1-2]; P < 0.0001) with increased time to first exacerbation (120 [87-161.5] d vs. 61.5 [20.7-122.7] d; P = 0.02). The gentamicin group had greater improvements in Leicester Cough Questionnaire (81.4% vs. 20%; P < 0.01) and St. George's Respiratory Questionnaire (87.5% vs. 19.2%; P < 0.004) score. No differences were seen in 24-hour sputum volume, FEV(1), FVC, or forced expiratory flow, midexpiratory phase. No P. aeruginosa isolates developed resistance to gentamicin. At follow-up, all outcome measures were similar to baseline. CONCLUSIONS: Regular, long-term nebulized gentamicin is of significant benefit in non-cystic fibrosis bronchiectasis but treatment needs to be continuous for its ongoing efficacy. Clinical trial registered with www.clinicaltrials.gov (NCT 00749866).


Assuntos
Antibacterianos/administração & dosagem , Bronquiectasia/tratamento farmacológico , Gentamicinas/administração & dosagem , Administração por Inalação , Aerossóis , Idoso , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória/métodos , Testes de Função Respiratória/estatística & dados numéricos , Cloreto de Sódio/administração & dosagem , Inquéritos e Questionários , Resultado do Tratamento
13.
Biochem Soc Trans ; 39(5): 1409-15, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21936824

RESUMO

WAP (whey acidic protein) is an important whey protein present in milk of mammals. This protein has characteristic domains, rich in cysteine residues, called 4-DSC (four-disulfide core domain). Other proteins, mainly present at mucosal surfaces, have been shown to also possess these characteristic WAP-4-DSC domains. The present review will focus on two WAP-4-DSC containing proteins, namely SLPI (secretory leucocyte protease inhibitor) and trappin-2/elafin. Although first described as antiproteases able to inhibit in particular host neutrophil proteases [NE (neutrophil elastase), cathepsin-G and proteinase-3] and as such, able to limit maladaptive tissue damage during inflammation, it has become apparent that these molecules have a variety of other functions (direct antimicrobial activity, bacterial opsonization, induction of adaptive immune responses, promotion of tissue repair, etc.). After providing information about the 'classical' antiproteasic role of these molecules, we will discuss the evidence pertaining to their pleiotropic functions in inflammation and immunity.


Assuntos
Elafina/imunologia , Imunidade nas Mucosas , Proteínas do Leite/química , Proteínas do Leite/imunologia , Inibidor Secretado de Peptidases Leucocitárias/imunologia , Animais , Elafina/química , Elafina/genética , Humanos , Inflamação/imunologia , Proteínas do Leite/genética , Inibidores de Proteases/química , Inibidores de Proteases/imunologia , Estrutura Terciária de Proteína , Inibidor Secretado de Peptidases Leucocitárias/química , Inibidor Secretado de Peptidases Leucocitárias/genética , Distribuição Tecidual
14.
Nat Commun ; 12(1): 765, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536414

RESUMO

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks.


Assuntos
Infecções por Escherichia coli/prevenção & controle , Escherichia coli/genética , Evolução Molecular , Genoma Bacteriano/genética , Doenças das Aves Domésticas/prevenção & controle , Animais , Galinhas , Escherichia coli/classificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Genes Bacterianos , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Humanos , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/microbiologia , Virulência/genética
15.
Am J Respir Cell Mol Biol ; 43(6): 692-702, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20097832

RESUMO

Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Brônquios/microbiologia , Brônquios/patologia , Epitélio/microbiologia , Epitélio/patologia , Infecções por Pseudomonas/patologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Brônquios/efeitos dos fármacos , Caspases/metabolismo , Comunicação Celular/efeitos dos fármacos , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Proteína X Associada a bcl-2/metabolismo , Catelicidinas
16.
Am J Respir Cell Mol Biol ; 43(1): 109-20, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19717812

RESUMO

Viral infections are known to exacerbate asthma and other lung diseases in which chronic inflammatory processes are implicated, but the mechanism is not well understood. The viral mimetic, polyinosine-polycytidylic acid, causes accumulation of a versican- and hyaluronan-enriched extracellular matrix (ECM) by human lung fibroblasts with increased capacity for monocyte adhesion. The fivefold increase in versican retention in this ECM is due to altered compartmentalization, with decreased degradation of cell layer-associated versican, rather than an increase in total accumulation in the culture. This is consistent with decreased mRNA levels for all of the versican splice variants. Reduced versican degradation is further supported by low levels of the epitope, DPEAAE, a product of versican digestion by a disintegrin-like and metallopeptidase with thrombospondin type 1 motif enzymes, in the ECM. The distribution of hyaluronan is similarly altered with a 3.5-fold increase in the cell layer. Pulse-chase studies of radiolabeled hyaluronan show a 50% reduction in the rate of loss from the cell layer over 24 hours. Formation of monocyte-retaining, hyaluronidase-sensitive ECMs can be blocked by the presence of anti-versican antibodies. In comparison, human lung fibroblasts treated with the cytokines, IL-1beta plus TNF-alpha, synthesize increased amounts of hyaluronan, but do not retain it or versican in the ECM, which, in turn, does not retain monocytes. These results highlight an important role for versican in the hyaluronan-dependent binding of monocytes to the ECM of lung fibroblasts stimulated with polyinosine-polycytidylic acid.


Assuntos
Matriz Extracelular/metabolismo , Monócitos/citologia , Poli C/metabolismo , Poli I/metabolismo , Versicanas/metabolismo , Adesão Celular , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/química , Humanos , Ácido Hialurônico/química , Inflamação , Interleucina-1beta/metabolismo , Pulmão/patologia , Monócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombospondinas/química , Trombospondinas/metabolismo
17.
Thorax ; 65(3): 201-7, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19825784

RESUMO

BACKGROUND: Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. METHODS: A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >10(4) colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from "non-VAP". Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. RESULTS: Seventy-two patients had recoverable lavage-24% had VAP. BALF interleukin-1beta (IL-1beta), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1alpha were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1beta generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1beta <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <10(4) cfu/ml. CONCLUSIONS: BALF IL-1beta and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.


Assuntos
Interleucina-1beta/análise , Interleucina-8/análise , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Métodos Epidemiológicos , Feminino , Humanos , Mediadores da Inflamação/análise , Masculino , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/microbiologia , Adulto Jovem
18.
Am J Pathol ; 174(4): 1338-46, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19264904

RESUMO

Microaspiration of Pseudomonas aeruginosa contributes to the pathogenesis of nosocomial pneumonia. Trappin-2 is a host defense peptide that assists with the clearance of P. aeruginosa through undefined mechanisms. A model of macrophage interactions with replicating P. aeruginosa (strain PA01) in serum-free conditions was developed, and the influence of subantimicrobial concentrations of trappin-2 was subsequently studied. PA01 that was pre-incubated with trappin-2 (at concentrations that have no direct antimicrobial effects), but not control PA01, was cleared by alveolar and bone marrow-derived macrophages. However, trappin-2-enhanced clearance of PA01 was completely abrogated by CD14- null macrophages. Fluorescence microscopy demonstrated the presence of trappin-2 on the bacterial cell surface of trappin-2-treated PA01. In a murine model of early lung infection, trappin-2-treated PA01 was cleared more efficiently than control PA01 2 hours of intratracheal instillation. Furthermore, trappin-2-treated PA01 up-regulated the murine chemokine CXCL1/KC after 2 hours with a corresponding increase in neutrophil recruitment 1 hour later. These in vivo trappin-2-treated PA01 effects were absent in CD14-deficient mice. Trappin-2 appears to opsonize P. aeruginosa for more efficient, CD14-dependent clearance by macrophages and contributes to the induction of chemokines that promote neutrophil recruitment. Trappin-2 may therefore play an important role in innate recognition and clearance of pathogens during the very earliest stages of pulmonary infection.


Assuntos
Elafina/imunologia , Receptores de Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Infiltração de Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Pneumopatias/imunologia , Pneumopatias/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose , Pseudomonas aeruginosa/imunologia , Proteínas Recombinantes/imunologia
20.
Am J Respir Crit Care Med ; 180(1): 19-28, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19324972

RESUMO

RATIONALE: Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood. OBJECTIVES: To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment. METHODS: Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions. MEASUREMENTS AND MAIN RESULTS: Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody. CONCLUSIONS: Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.


Assuntos
Complemento C5a/fisiologia , Neutrófilos/fisiologia , Fagocitose/fisiologia , Pneumonia Associada à Ventilação Mecânica/sangue , Receptores de Complemento/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Estudos de Casos e Controles , Complemento C3a/análise , Complemento C3a/fisiologia , Complemento C5a/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/imunologia , Receptor da Anafilatoxina C5a
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