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BACKGROUND: Studies preceding the COVID-19 pandemic found that slower time-to-return was associated with first-time, deferred, and mobile drive blood donors. How donor return dynamics changed during the COVID-19 pandemic is not well understood. METHODS: We analyzed visits by whole blood donors from 2017 to 2022 in South Africa (SA) and the United States (US) stratified by mobile and fixed environment, first-time and repeat donor status, and pre-COVID19 (before March 2020) and intra-COVID19. We used Kaplan-Meier curves to characterize time-to-return, cumulative incidence functions to analyze switching between donation environments, and Cox proportional hazards models to analyze factors influencing time-to-return. RESULTS: Overall time-to-return was shorter in SA. Pre-COVID19, the proportion of donors returning within a year of becoming eligible was lower for deferred donors in both countries regardless of donation environment and deferral type. Intra-COVID19, the gap between deferred and non-deferred donors widened in the US but narrowed in SA, where efforts to schedule return visits from deferred donors were intensified, particularly for non-hemoglobin-related deferrals. Intra-COVID19, the proportion of donors returning within a year in SA was higher for deferred first-time donors (>81%) than for successful first-time donors (80% at fixed sites; 69% at mobile drives). CONCLUSIONS: The pandemic complicated efforts to recruit new donors and schedule returning visits after completed donations. Concerted efforts to improve time-to-return for deferred donors helped mitigate donation loss in SA during the public health emergency.
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Doadores de Sangue , COVID-19 , SARS-CoV-2 , Humanos , Doadores de Sangue/estatística & dados numéricos , COVID-19/epidemiologia , África do Sul/epidemiologia , Estados Unidos/epidemiologia , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , PandemiasRESUMO
The current study evaluated the potential of clinical parameters and circulating biomarkers to distinguish sepsis from SIRS in patients admitted with systemic inflammation. Clinical parameters, leukocyte counts and platelets were measured on admission. Circulating C-reactive protein (CRP), procalcitonin (PCT) and cytokine concentrations were quantified using laser immunonephelometry, immunoluminescence and a Bio-Plex suspension bead array system respectively. Blood, sputum, urine, peritoneal and cerebrospinal fluid were sent for microscopy and culture. Based on clinical information and the results of microbiological testing, 62 patients were classified retrospectively into 2 groups, those with sepsis (nâ¯=â¯37) or SIRS (nâ¯=â¯25). Mean body temperature was higher and blood pressure lower in the sepsis patients. Circulating concentrations of CRP, PCT, interleukin (IL)-10 and IL-1 receptor antagonist (IL-1Ra) were significantly higher in patients with sepsis, with IL-10 identified as the best biomarker in differentiating sepsis from SIRS. The biomarkers that best predicted overall mortality were platelet counts >PCTâ¯≥â¯CRPâ¯>â¯IL-6â¯>â¯IL-1Ra. These findings demonstrate that patients with sepsis have significantly increased levels of the immunosuppressive/anti-inflammatory cytokines, IL-1Ra and IL-10, compared to those with SIRS, consistent with a more intense counteracting anti-inflammatory response, while a biomarker profile including platelets, PCT, CRP, IL-6 and IL-1Ra may be useful to predict mortality.
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Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-10/sangue , Sepse/sangue , Sepse/diagnóstico , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Adulto , Biomarcadores/sangue , Diagnóstico Diferencial , Feminino , Humanos , Inflamação/sangue , Masculino , Valor Preditivo dos Testes , Curva ROC , Sensibilidade e Especificidade , Sepse/microbiologia , Sepse/mortalidade , Sobreviventes , Síndrome de Resposta Inflamatória Sistêmica/mortalidadeRESUMO
Although the well-recognised predisposition of cigarette smokers to the development of severe pneumococcal disease may be attributable to impairment of local host defences, less is known about the direct effects of smoke exposure on airway pathogens, or their virulence factors. In the current study, we have investigated the effects of cigarette smoke condensate (CSC) on biofilm formation by Streptococcus pneumoniae, and on the pore-forming activity of its major toxin, pneumolysin. Biofilm formation following exposure of the pneumococcus to CSC (20-160 µg·mL(-1)) was measured using a crystal violet-based spectrophotometric procedure, while the pore-forming activity of recombinant pneumolysin was determined by a fura-2/acetoxymethyl ester-based spectrofluorimetric procedure to monitor the uptake of extracellular Ca(2+) by isolated human neutrophils. Exposure of the pneumococcus or pneumolysin to CSC resulted in significant dose-related augmentation of biofilm formation (p≤0.05 at 80 and 160 µg·mL(-1)) and substantial attenuation of the pore-forming interactions of pneumolysin, respectively. Augmentation of biofilm formation and inactivation of pneumolysin as a consequence of smoking are likely to favour microbial colonisation and persistence, both being essential precursors of pneumococcal disease.
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Biofilmes , Neutrófilos/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Estreptolisinas/biossíntese , Proteínas de Bactérias/biossíntese , Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Corantes Fluorescentes , Fura-2 , Humanos , Neutrófilos/metabolismo , Fumar/efeitos adversos , Espectrometria de FluorescênciaRESUMO
Community-acquired pneumonia (CAP) remains a leading cause of morbidity and mortality among the infectious diseases. Despite the implementation of national pneumococcal polyvalent vaccine-based immunisation strategies targeted at high-risk groups, Streptococcus pneumoniae (the pneumococcus) remains the most common cause of CAP. Notwithstanding the HIV pandemic, major challenges confronting the control of CAP include the range of bacterial and viral pathogens causing this condition, the ever-increasing problem of antibiotic resistance worldwide, and increased vulnerability associated with steadily aging populations in developed countries. These and other risk factors, as well as diagnostic strategies, are covered in the first section of this review. Thereafter, the review is focused on the pneumococcus, specifically the major virulence factors of this microbial pathogen and their role in triggering overexuberant inflammatory responses which contribute to the immunopathogenesis of invasive disease. The final section of the review is devoted to a consideration of pharmacological, anti-inflammatory strategies with adjunctive potential in the antimicrobial chemotherapy of CAP. This is focused on macrolides, corticosteroids, and statins with respect to their modes of anti-inflammatory action, current status, and limitations.
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Infecções Comunitárias Adquiridas/etiologia , Inflamação/complicações , Pneumonia Bacteriana/etiologia , Biofilmes , Infecções Comunitárias Adquiridas/tratamento farmacológico , Infecções Comunitárias Adquiridas/imunologia , Infecções Comunitárias Adquiridas/mortalidade , Efeitos Psicossociais da Doença , Farmacorresistência Bacteriana , Humanos , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/mortalidade , Pneumonia Pneumocócica/etiologia , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de DoençaRESUMO
OBJECTIVES: Tigecycline is the prototype of the recently introduced, intravenously administered glycylcycline class of antibiotics, developed in response to the increasing problem of antibiotic resistance in Gram-positive bacteria, especially Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. However, relatively little is known about the immunomodulatory potential of tigecycline, specifically its interactions with human neutrophils. In the current study we investigated the effects of tigecycline at therapeutically relevant concentrations and greater (0.625-10 mg/L) on alterations in cytosolic Ca(2+) concentrations, generation of antimicrobial reactive oxygen species (ROS) and release of granule proteases [elastase, matrix metalloproteinase-8 (MMP-8) and matrix metalloproteinase-9 (MMP-9)] by human blood neutrophils activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 µM). METHODS: Cytosolic Ca(2+) concentrations were measured using fura-2/AM-based spectrofluorimetry and radiometric procedures, generation of ROS by oxygen consumption and myeloperoxidase-mediated auto-iodination, and protease release by ELISA procedures. RESULTS: Exposure of the cells to fMLP resulted in activation of the generation of ROS, as well as release of the granule proteases, all of which were significantly increased by pre-incubation of the cells with tigecycline in a dose-dependent manner. Tigecycline-mediated enhancement of these neutrophil functions was associated with elevations in the concentrations of cytosolic Ca(2+), which appeared to result from the Ca(2+) ionophore activity of tigecycline. CONCLUSIONS: Tigecycline, by functioning as a Ca(2+) ionophore, and independent of antimicrobial activity, potentiates the pro-inflammatory functions of human neutrophils in vitro.
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Cálcio/metabolismo , Fatores Imunológicos/metabolismo , Minociclina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Adulto , Células Cultivadas , Citosol/química , Experimentação Humana , Humanos , Minociclina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/imunologia , TigeciclinaRESUMO
Macrolide antibiotics possess several, beneficial, secondary properties which complement their primary antimicrobial activity. In addition to high levels of tissue penetration, which may counteract seemingly macrolide-resistant bacterial pathogens, these agents also possess anti-inflammatory properties, unrelated to their primary antimicrobial activity. Macrolides target cells of both the innate and adaptive immune systems, as well as structural cells, and are beneficial in controlling harmful inflammatory responses during acute and chronic bacterial infection. These secondary anti-inflammatory activities of macrolides appear to be particularly effective in attenuating neutrophil-mediated inflammation. This, in turn, may contribute to the usefulness of these agents in the treatment of acute and chronic inflammatory disorders of both microbial and nonmicrobial origin, predominantly of the airways. This paper is focused on the various mechanisms of macrolide-mediated anti-inflammatory activity which target both microbial pathogens and the cells of the innate and adaptive immune systems, with emphasis on their clinical relevance.
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Antibacterianos/farmacocinética , Antibacterianos/uso terapêutico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Imunidade Adaptativa/efeitos dos fármacos , Animais , Humanos , Imunidade Inata/efeitos dos fármacos , Fagócitos/efeitos dos fármacosRESUMO
Streptococcus pneumoniae is a leading cause of pneumonia mortality globally. Pneumococcal disease is often associated with prolonged colonisation of hosts and this process is facilitated by biofilm formation that is largely resistant to conventional antibiotics. We investigated the effects of antimicrobial peptides (AMPs) lysozyme, lactoferrin, LL37 and a combination of all three on planktonic growth, biofilm formation and biofilm-derived bacterial viability by S. pneumoniae, serotype 23F. Planktonic growth and biofilm-derived bacterial viability were determined using standard colony-forming techniques, while biofilm formation was measured using a crystal violet based spectrophotometric method. Relative to controls, lysozyme significantly reduced biofilm formation (0.08 OD vs. 0.10 OD at 570 nm, p = 0.01), while LL37 and the AMP combination increased biofilm formation (0.14 OD vs. 0.10 OD at 570 nm, p = 0.01). The combination of AMPs significantly decreased planktonic growth (1.10 × 108 colony-forming units per millilitres [CFU/mL] vs. 2.13 × 108 CFU/mL, p = 0.02). Biofilm-derived bacterial viability was greatly reduced by exposure to a combination of AMPs (1.05 × 105 CFU/mL vs. 1.12 × 106 CFU/mL, p = 3.60 × 10-8). Streptococcus pneumoniae displays marked resistance to the individual AMPs. A combination of lysozyme, lactoferrin and LL37 effectively inhibited planktonic growth and biofilm-derived bacterial viability; however, persister cell growth was still evident after exposure.
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OBJECTIVES: Exposure to cigarette smoke impacts on the virulence of Streptococcus pneumoniae (pneumococcus) by mechanisms including induction of biofilm formation. Most studies, however, have focused on individual strains of the pneumococcus. Accordingly, the current study has investigated the commonality of the pneumococcal stress response to cigarette smoke condensate (CSC), using five different strains of the pathogen. METHODS: Following exposure to CSC at final concentrations of 80 and 160⯵g mL-1 during a 16 h incubation period, biofilm formation was measured using a crystal violet-based spectrophotometric procedure. Expression of stress genes seemingly linked to biofilm formation viz. hk11 and rr11 [histidine kinase and response regulator of the two-component regulatory system 11 (TCS11) respectively], cat eff (cation efflux system protein), abc (ATP-binding component of an ATP-binding cassette transporter) and 2005-hyp (hypothetical gene) was measured by sequential extraction of RNA, cDNA synthesis and real-time qPCR. RESULTS: Exposure of all five strains of the pneumococcus to CSC, resulted in significant biofilm formation, as well as induction of all five test stress genes. CONCLUSIONS: Augmentation of induction of selective stress genes and biofilm formation are common, possibly linked, responses of various serotypes of the pneumococcus to CSC, favouring both persistence of the pathogen and decreased efficacy of antibiotics.
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Biofilmes , Fumaça/efeitos adversos , Streptococcus pneumoniae , Humanos , Sorogrupo , Fumar , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiologiaRESUMO
BACKGROUND: The role of the oral mucosa as a target of human immunodeficiency virus (HIV-1) infection and persistence is unclear. HIV-1 has been reported in oral epithelial cells, but this has not been confirmed. Cellular reservoirs may impede antiretroviral therapies and should be identified. This study was performed to determine the presence of HIV-1 in oral epithelial and Langerhans cells (LCs) of HIV-1-positive antiretroviral naïve patients. Non-invasive brush biopsy technique for future in vivo HIV research was also evaluated. METHODS: Oral mucosal cells were harvested from the buccal mucosae, dorsal tongue and the gingiva of the mandibular teeth of 35 HIV-1-positive patients using a Cytobrush Plus cell collector. Epithelial cells were purified from the samples by flow cytometric cell sorting using cytokeratin stains after which the epithelial cell samples were further purified and divided into superficial and deep epithelial cells by laser microdissection on Pap stained cytospin smears. LCs were picked up individually by laser microdissection from CD1a stained cytospin smears. Purified epithelial and LC samples were tested for the presence of HIV-1 DNA by polymerase chain reaction analysis. RESULTS: Ten of the patients had HIV-1 DNA in one or more of the sampled anatomical locations. No HIV-1 DNA could be demonstrated in any of the purified superficial or deep epithelial or LC samples. CONCLUSIONS: HIV-DNA can be found using non-invasive oral brush biopsies and should be investigated further as an experimental model for in vivo oral HIV research. Better ways to purify the different cell types should be investigated.
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HIV-1/fisiologia , Células de Langerhans/virologia , Mucosa Bucal/virologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Adolescente , Adulto , Paralisia de Bell/virologia , Contagem de Linfócito CD4 , Candidíase Bucal/virologia , Citodiagnóstico/instrumentação , Citodiagnóstico/métodos , DNA Viral/análise , Cárie Dentária/virologia , Reservatórios de Doenças/virologia , Células Epiteliais/virologia , Feminino , Gengiva/patologia , Gengiva/virologia , Glossite/virologia , Soropositividade para HIV/virologia , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/patologia , Periodontite/virologia , Língua/patologia , Língua/virologia , Carga Viral , Latência Viral , Adulto JovemRESUMO
INTRODUCTION: Cigarette smoking is a well-recognized risk factor for development of severe, invasive pneumococcal disease. However, little is known about the direct effects of exposure to cigarette smoke on the virulence mechanisms of the pathogen, particularly in respect of resistance to macrolide antibiotics, which are widely used in the treatment of pneumococcal infection. This study aimed to investigate the effects of exposure to cigarette smoke condensate (CSC, 80 and 160 mg/L) and clarithromycin (2 and 8 mg/L), alone and in combination in vitro, on expression of the erm(B) and mef(A) macrolide resistance genes of strains 2507 and 521 (both serotype 23F), respectively, of the pneumococcus. METHODS: Following exposure to CSC or clarithromycin, individually and in combination, erm(B) and mef(A) gene expression were measured by sequential extraction of RNA, conversion to and amplification of cDNA, and detection by qRT-PCR. RESULTS: As expected, exposure of both test strains of the pneumococcus to clarithromycin resulted in substantial upregulation of both macrolide resistance genes, which was significantly (p<0.001) augmented by prior exposure to CSC in the case of erm(B), but not mef(A). Somewhat unexpectedly, exposure of strain 2507 to CSC (160 mg/L) alone (in the absence of clarithromycin) also resulted in significant (p<0.05) expression of the erm(B) gene. CONCLUSION: Although the possible clinical significance remains to be established, these findings suggest that smoking may impede the efficacy of macrolide-based antimicrobial therapy by accelerating the onset and magnitude of erm(B)-mediated resistance, representing a novel pro-infective mechanism of smoking.
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Streptococcus pneumoniae (the pneumococcus) remains one of the major human pathogens and one of the most common causes of community-acquired pneumonia, otitis media, sinusitis, and meningitis. Aside from the threats posed by emerging antibiotic resistance and infection with the human immunodeficiency virus, the mortality rate among those patients with severe pneumococcal disease who receive seemingly appropriate antimicrobial chemotherapy remains unacceptably high. Because of its involvement in the pathogenesis of invasive disease, pneumolysin, one of the best-characterized virulence factors of the pneumococcus, represents not only a potential vaccine target, but also a target for adjunctive therapy to antibiotics in patients with acute pneumococcal disease. In this paper we review the cytolytic and pro-inflammatory properties of pneumolysin and their involvement in subversion of host defenses and extra-pulmonary dissemination of the pneumococcus, as well as strategies, both immunological and pharmacological, which may counter these harmful activities of the toxin.
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Infecções Pneumocócicas/tratamento farmacológico , Pneumonia Pneumocócica/tratamento farmacológico , Streptococcus pneumoniae/patogenicidade , Estreptolisinas/farmacologia , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Proteínas de Bactérias/farmacologia , Humanos , Modelos Biológicos , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/farmacologia , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/prevenção & controle , Fatores de Risco , Vacinas Conjugadas/farmacologiaRESUMO
Recent insights into the immunopathogenesis of pneumococcal infection, a common and significant cause of morbidity and mortality, have implicated pneumolysin as being a prominent virulence factor, which may play a role in microbial colonization, invasion and dissemination, as well as tissue inflammation. Being a highly immunogenic polypeptide produced by all clinically relevant pneumococcal isolates, pneumolysin is recognized as a potential carrier protein for polysaccharide conjugate vaccines, while in the setting of acute disease, promising pneumolysin-directed pharmacological strategies include, among others, macrolides and corticosteroids.
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Proteínas de Bactérias/fisiologia , Infecções Pneumocócicas/fisiopatologia , Estreptolisinas/fisiologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Humanos , Imunização , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/prevenção & controle , Fatores de Risco , Estreptolisinas/antagonistas & inibidoresRESUMO
This study was designed to investigate the effects of the Streptococcus pneumoniae-derived, pro-inflammatory toxin, pneumolysin (8.37 and 41.75 ng/ml), on the oxidative inactivation of alpha-1-protease inhibitor (API) by chemoattractant-activated human neutrophils in vitro. The elastase inhibitory capacity (EIC) of API in supernatants from unstimulated neutrophils, neutrophils treated with pneumolysin only, or with the chemoattractant FMLP (1 microM) only, or the combination of the toxin with FMLP was measured by a colorimetric procedure based on the activity of added porcine elastase. The EIC of API was unaffected by exposure to pneumolysin only, unstimulated neutrophils, or neutrophils treated with pneumolysin only. However, exposure to FMLP-activated neutrophils resulted in a reduction of the EIC of API, which was significantly (P<0.05) augmented by pneumolysin (mean reductions of 16%, 43% and 83% for FMLP only and in combination with 8.37 and 41.75 ng/ml pneumolysin, respectively), and was attenuated by wortmannin (1 microM), an inhibitor of NADPH oxidase, the oxidant-scavenger methionine (100 microM), and depletion of Ca2+ from the cell-suspending medium. These pro-proteolytic interactions of pneumolysin with chemoattractant-activated neutrophils may contribute to the invasiveness of the pneumococcus.
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Neutrófilos/efeitos dos fármacos , Estreptolisinas/farmacologia , Inibidores da Tripsina/farmacologia , alfa 1-Antitripsina/imunologia , Adulto , Androstadienos/imunologia , Androstadienos/farmacologia , Proteínas de Bactérias , Quimiotaxia de Leucócito/imunologia , Humanos , Elastase de Leucócito/imunologia , Metionina/imunologia , N-Formilmetionina Leucil-Fenilalanina , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Oxirredução , Fosfatidilinositol 3-Quinases/imunologia , Estreptolisinas/imunologia , Superóxidos/imunologia , Inibidores da Tripsina/imunologia , WortmaninaRESUMO
Catheter-related blood stream infections increase morbidity, mortality, and costs. This study investigated whether Certofix(®) protect antimicrobial catheters carry a surface charge and whether this inhibits biofilm formation. The capacitance of the catheter surfaces was measured and, to determine if the catheters released ions, distilled water was passed through and current measured as a function of voltage. With probes touching the inner and outer surfaces, capacitance was not voltage-dependent, indicating surfaces were uncharged or carried a similar charge. When one probe penetrated the catheter wall, capacitance was weakly voltage-dependent, indicating the presence of a surface charge. Standard and charged catheters were also exposed to phosphate buffered saline as controls or 2×10(6) colony forming units/mL (in phosphate buffered saline) of six different microorganisms for 60 or 120 minutes. When the growth of detached bacteria was measured, biofilm formation was significantly reduced, (P<0.05), for charged catheters for all organisms.
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Alterations in whole genome expression profiles following exposure of the pneumococcus (strain 172, serotype 23F) to cigarette smoke condensate (160 µg/mL) for 15 and 60 min have been determined using the TIGR4 DNA microarray chip. Exposure to CSC resulted in the significant (P<0.014-0.0006) upregulation of the genes encoding the two-component regulatory system 11 (TCS11), consisting of the sensor kinase, hk11, and its cognate response regulator, rr11, in the setting of increased biofilm formation. These effects of cigarette smoke on the pneumococcus may contribute to colonization of the airways by this microbial pathogen.
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Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Membrana/biossíntese , Proteínas Quinases/biossíntese , Fumar/efeitos adversos , Streptococcus pneumoniae/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Histidina Quinase , Análise de Sequência com Séries de Oligonucleotídeos , Sorogrupo , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
In general antibiotics interact cooperatively with host defences, weakening and decreasing the virulence of microbial pathogens, thereby increasing vulnerability to phagocytosis and eradication by the intrinsic antimicrobial systems of the host. Antibiotics, however, also interact with host defences by several other mechanisms, some harmful, others beneficial. Harmful activities include exacerbation of potentially damaging inflammatory responses, a property of cell-wall targeted agents, which promotes the release of pro-inflammatory microbial cytotoxins and cell-wall components. On the other hand, inhibitors of bacterial protein synthesis, especially macrolides, possess beneficial anti-inflammatory/cytoprotective activities, which result from interference with the production of microbial virulence factors/cytotoxins. In addition to these pathogen-directed, anti-inflammatory activities, some classes of antimicrobial agent possess secondary anti-inflammatory properties, unrelated to their conventional antimicrobial activities, which target cells of the innate immune system, particularly neutrophils. This is a relatively uncommon, potentially beneficial property of antibiotics, which has been described for macrolides, imidazole anti-mycotics, fluoroquinolones, and tetracyclines. Although of largely unproven significance in the clinical setting, increasing awareness of the pro-inflammatory and anti-inflammatory properties of antibiotics may contribute to a more discerning and effective use of these agents.
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BACKGROUND: The role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM), was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 muM) and staurosporine (400 nM). METHODS: Alterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3), and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively. RESULTS: Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase.Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils. CONCLUSION: Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.
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In addition to being cytotoxic for eukaryotic cells, recent research has clearly indicated that pneumolysin at sub-cytolytic concentrations potentiates the proinflammatory activities of neutrophils and macrophages. Together these cytotoxic and proinflammatory activities of the toxin are likely to contribute to the virulence of the pneumococcus, particularly in facilitating adherence, invasion and dissemination of this important microbial pathogen. Pneumolysin-based vaccine strategies, although in the early stages of development and evaluation, show promise in reducing the severity of pneumococcal disease.
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Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/patogenicidade , Estreptolisinas/imunologia , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Infecções Pneumocócicas/diagnóstico , Vacinas Pneumocócicas/química , Vacinas Pneumocócicas/imunologia , Conformação Proteica , Streptococcus pneumoniae/fisiologia , Estreptolisinas/química , Estreptolisinas/toxicidade , VirulênciaRESUMO
Pneumolysin (4.18 ng/ml)-mediated influx of Ca(2+) and augmentation of the chemoattractant-activated generation of reactive oxidants was antagonized by pretreatment of human neutrophils with the omega-3 polyunsaturated fatty acids docosahexaenoic acid and eicosapentaenoic acid (1.25 to 5 microg/ml). These agents may have potential in attenuating the proinflammatory properties of this pneumococcal toxin.
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Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Inflamação/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Estreptolisinas/metabolismo , Proteínas de Bactérias , Cálcio/metabolismo , Humanos , Neutrófilos/metabolismo , Oxidantes/metabolismoRESUMO
The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 and 41.75 ng/mL), has on the production of interleukin (IL)-8 and tumor-necrosis factor (TNF)-alpha by human neutrophils have been investigated in vitro. Total and extracellular IL-8 and TNF-alpha were assayed by enzyme-linked immunosorbent assay, and flow cytometry and colorimetric procedures were used to detect intracellular cytokine and cytokine messenger RNA, respectively. Treatment of neutrophils with pneumolysin either alone or in combination with the chemotactic tripeptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), resulted in a time-dependent (maximal at 6 h) increase in synthesis and release of IL-8 but not of TNF-alpha, which was associated with increased expression of IL-8 messenger RNA transcripts and was abrogated by either cycloheximide (10 microg/mL) or depletion of Ca(2+) from the cell-suspending medium. These interactions between the toxin and neutrophils may contribute to the exaggerated pulmonary inflammatory responses caused by pneumolysin-producing strains of the pneumococcus.