RESUMO
BACKGROUND: Increased antibiotic resistant properties of pathogens has led to a pronounced search for new effective antibiotics from microbes in diverse ecological niches. This study focused on isolating actinomycetes from soil of reserved areas and profiling them for antibacterial potentials. METHODS: The isolates (IS-2, IS-4, IS-6, IS-10, IS-14) were assessed for antagonistic activity against ten multi-drug resistant bacterial strains (Gram positive and negative) by cross streak and well diffusion methods. RESULTS: During the primary screening, four of the isolates showed good antagonistic activity against the test strains. Notably, all the bacterial strains except Pseudomonas aeruginosa responded sensitively to at least one of the actinomycetes screened. The crude extracts of the secondary metabolites of the four actinomycetes (IS-2, IS-4, IS-6, IS-10) with considerably significant antagonistic activities inhibited the growth of all the bacterial strains efficiently. All the test bacterial strains were sensitive to at least one of the extracts at a concentration of 100µg/ml. The minimum inhibitory concentration of the extracts against the isolates ranged from 12.5 - 25µg/ml. The crude extracts of IS-4 and IS-6 identified as Streptomyces glauciniger NBRC 100913 and Streptomyces griseoplanus NRRL-ISP 5009 by I6s rRNA sequencing, showed higher antibacterial activities against the bacterial strains. Significantly, the ethyl acetate crude extract of the actinomycetes demonstrated better antibacterial activities than the standard antibiotics (ofloxacin, amoxicillin/clavulanate, cefuroxime and ceftriaxone). CONCLUSION: This study reports remarkable anti-MRSA activities as well as broad spectrum antibacterial potentials of extracts of Streptomyces spp. worthy of further exploration.
Assuntos
Actinobacteria/fisiologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Microbiologia do Solo , Células Cultivadas/microbiologia , NigériaRESUMO
This Article describes a density-based method for removing contaminants, including microorganisms and nonviable cells, from mammalian cell cultures using an aqueous two-phase system (ATPS). The properties of a 7% w/w polyethylene glycol (PEG)-11% w/w Ficoll ATPS can be tuned to prepare a biocompatible system that removes contaminants with little to no adverse effects on the viability or growth of the cultured cells after treatment. This system can be used to enrich cell culture populations for viable cells and to reduce the number of microorganism contaminants in a culture, which increases the chances of subsequent antibiotic treatments being successful. We test the effectiveness of our method in model contaminated cultures of both adherent (HeLa) and suspension (HL-60 II) mammalian cells contaminated with bacteria (E. coli) and yeast (S. cerevisiae). An average of 70.2 ± 4.6% of HeLa cells added to the system are subsequently recovered, and 55.9 ± 2.1% of HL-60 II cells are recovered. After sedimenting to the interface of the ATPS, these cells have an average viability of 98.0 ± 0.2% and 95.3 ± 2.2%, respectively. By removing unwanted cells, desired cell populations can be recovered, and cultures that would otherwise need to be discarded can continue to be used.
Assuntos
Células Cultivadas/microbiologia , Meios de Cultura/isolamento & purificação , Contaminação de Equipamentos/prevenção & controle , Extração em Fase Sólida/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Centrifugação/métodos , Escherichia coli/isolamento & purificação , Ficoll/química , Humanos , Fenômenos Físicos , Polietilenoglicóis/química , Saccharomyces cerevisiae/isolamento & purificação , Água/químicaRESUMO
A variety of biological and chemical contaminants can adversely impact cells in culture, ranging from outright destruction of the culture, mutation, phenotypic changes to relatively minor changes in morphology, or growth rate. There are various approaches to detecting and mitigating the risk of biological or microbial contaminants in cell cultures, and these are discussed in this article. Chemical contaminants typically arise from improper handling or sourcing of cell culture reagents, glassware, or other types of consumables. These and other sources of chemical contaminants of cell cultures are discussed. The occurrence of chemical contamination is mitigated through adherence to best practices in sourcing and handling of such materials and by avoiding the use of volatile solvents within incubators that are used for maintaining cell cultures.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas Microbiológicas/métodos , Animais , Antibacterianos/farmacologia , Células Cultivadas/microbiologia , Meios de Cultura/análise , Meios de Cultura/química , Detergentes , Endotoxinas , Radicais Livres , Humanos , Metais PesadosRESUMO
Fungal contamination of in vitro cultures of date palm (Phoenix dactylifera L.) is the major constraint to their initiation and maintenance. Different molecular approaches have been applied successfully to analyze both inter- and intraspecific variation among fungal species as well as determine their identity. This chapter describes step-by-step procedures of molecular identification of fungal contaminants by internal transcribed spacer (ITS) products of the most common fungal contaminants of date palm tissue culture. To begin with, samples of genera Alternaria, Aspergillus, Cladosporium, Epicoccum, and Penicillium were collected to isolate each fungal genus and extraction of genomic DNA. Polymerase chain reactions were accomplished by ITS primers (ITS1 and ITS4) for each fungal contaminant as well as for sequencing. Subsequently, they are analyzed by Basic Local Alignment Search Tool (BLAST) search of ITS sequence to reveal the identity of each individual fungal contaminant species. The molecular identification herein is a rapid and reliable procedure to identify date palm fungal contaminants which is very important in their control and treatment.
Assuntos
Células Cultivadas/microbiologia , Fungos/genética , Phoeniceae/microbiologia , DNA Fúngico/genética , Reação em Cadeia da Polimerase/métodos , Técnicas de Cultura de Tecidos/métodosRESUMO
The aim of this study was to evaluate the role of microscopy, Gram stain and the culture of tissue samples in the antibiotic treatment of patients with diabetic foot infection. A retrospective review of patients with a diabetic foot infection was undertaken. Data analysed included the severity of infection, antibiotic prescribing patterns, microscopy and culture results. A total of 71 patients were included, from whom 114 tissue samples were collected. Gram stain results were in agreement with final culture results in 45·8% (n = 54) of samples. Overall sensitivity and specificity of the Gram stains were low (74·5% and 69·8%, respectively), although the specificity for Gram-negative rods was high (98·5%). The presence or absence of 'pus cells' on microscopy was a poor predictor of culture results. Empirical prescribing of antibiotics was in accordance with local policy in 31·1% of patients, improving to 86·8 % following culture results. Microscopy, a skilled laboratory procedure, was generally a poor predictor of tissue culture results. However, the presence of Gram-negative rods was suggestive of isolation in the culture of such organisms and could allow the early broadening of antibiotic treatment. Despite initial poor compliance of empirical antibiotic treatment regimens, prescribing was adjusted in light of culture results, suggesting these were important for clinicians.
Assuntos
Antibacterianos/uso terapêutico , Células Cultivadas/microbiologia , Pé Diabético/tratamento farmacológico , Pé Diabético/microbiologia , Bactérias Gram-Negativas/isolamento & purificação , Microscopia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Violeta Genciana , Humanos , Masculino , Pessoa de Meia-Idade , Fenazinas , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the pro-inflammatory cytokine, IL-1ß, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs.
Assuntos
Proteína HMGB1/metabolismo , Inflamassomos/imunologia , Núcleosídeo-Difosfato Quinase/metabolismo , Porphyromonas gingivalis/imunologia , Trifosfato de Adenosina/metabolismo , Células Cultivadas/microbiologia , Gengiva/citologia , Proteína HMGB1/imunologia , Humanos , Inflamassomos/metabolismo , Núcleosídeo-Difosfato Quinase/imunologia , Porphyromonas gingivalis/patogenicidade , Transdução de Sinais/efeitos dos fármacosRESUMO
Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as ≥100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. This was unexpected given 37% of contaminated series used poly(A)-selection for mRNA enrichment. Lastly, we examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality.
Assuntos
Bases de Dados de Ácidos Nucleicos , Mycoplasma/genética , RNA/genética , Transcriptoma/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas/microbiologia , Biologia Computacional/métodos , Interações Hospedeiro-Patógeno/genética , Humanos , Mycoplasma/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , RNA Bacteriano/genética , Medição de Risco/métodos , Medição de Risco/estatística & dados numéricos , Alinhamento de Sequência/métodosAssuntos
Células Cultivadas/microbiologia , Obtenção de Tecidos e Órgãos/normas , Animais , Pesquisa Biomédica , Células Cultivadas/virologia , Engenharia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Guias como Assunto , Humanos , Itália , Laboratórios , Mycoplasma/isolamento & purificação , Príons/isolamento & purificação , Príons/patogenicidade , Medição de Risco , Fatores de Risco , Células Tumorais Cultivadas , Proteínas Virais/efeitos adversos , Latência ViralRESUMO
BACKGROUND AND OBJECTIVE: Aminopeptidase N (CD13) is an ectoenzyme located in the outer membrane of a variety of cells. Proteomic profiling indicates an increased expression of CD13 in phagocytes during Mycobacterium tuberculosis infection. The purpose of this study was to investigate the role of CD13 on the internalization and intracellular survival of M. tuberculosis in monocytes. METHODS: Magnetic nanoparticles and confocal microscopy were used to observe interactions between CD13 and M. tuberculosis. Mycobacterial entry and intracellular survival in monocytes were assessed with and without anti-CD13 antibody (WM15 and WM47) using flow cytometry and colony formation assay. RESULTS: By using magnetic nanoparticles and confocal microscopy, M. tuberculosis was found to be capable of binding to either soluble CD13 or membranous CD13 on monocytes. Flow cytometry showed that pretreatment of monocytes with WM15 or WM47 reduced the number of intracellular M. tuberculosis. Collectively, the data suggest that CD13 is a binding and entry receptor for M. tuberculosis on monocytes. Treatment of infected monocytes showed a greater effect of WM47 than WM15 in reducing the intracellular colonization of M. tuberculosis, suggesting that specific epitopes of CD13 may play an important role modulating intracellular M. tuberculosis survival. CONCLUSIONS: CD13 acts as a receptor for M. tuberculosis on human monocytes. The molecule facilitates internalization, and interaction of CD13 with an anti-CD13 antibody reduces intracellular M. tuberculosis survival.
Assuntos
Antígenos CD13/metabolismo , Monócitos/enzimologia , Mycobacterium tuberculosis/enzimologia , Tuberculose/microbiologia , Células Cultivadas/microbiologia , Citometria de Fluxo , Humanos , Microscopia Confocal , Mycobacterium tuberculosis/isolamento & purificação , Proteômica/métodos , Tuberculose/enzimologia , Tuberculose/patologiaRESUMO
Yersinia enterocolitica biovar 1A strains have been delineated into two clonal groups (A and B) based on repetitive extragenic palindrome- and enterobacterial repetitive intergenic consensus-PCR genotyping. The present study investigated the interaction of Y. enterocolitica biovar 1A strains with cultured cells in vitro by their ability to adhere, invade and survive within these cells. The response of macrophages to these strains was also studied by quantifying the expression of inducible nitric oxide synthase, production of nitric oxide and cytokines, and activation of NFκB. The survival rate of clonal group B strains inside macrophages was significantly higher than that of clonal group A strains. In addition, strains harbouring the fepA gene showed better survival inside macrophages. However, the production of nitric oxide and cytokines and activation of NFκB did not show any significant differences between the two clonal groups. In this study, interaction of Y. enterocolitica biovar 1A with cultured cells in vitro did not reflect the previously identified clonal groups, but was more dependent on the characteristics of the individual strains. Therefore, a combination of genotype and phenotype data must be used to characterize this extremely heterogeneous organism.
Assuntos
Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Yersinia enterocolitica/metabolismo , Animais , Adesão Celular/genética , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Genótipo , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Taxa de Sobrevida , Yersinia enterocolitica/genéticaRESUMO
Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the haemolymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were continuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. However, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis.
Assuntos
Ehrlichia/genética , Ehrlichia/isolamento & purificação , Ehrlichiose/microbiologia , RNA Ribossômico 16S/genética , Rhipicephalus/microbiologia , Animais , Brasil , Bovinos , Células Cultivadas/microbiologia , Cães , Ehrlichiose/transmissão , Ehrlichiose/veterinária , Feminino , Genótipo , Filogenia , Reação em Cadeia da Polimerase , Carrapatos/microbiologiaRESUMO
Anaplasma phagocytophilum, first identified as a pathogen of sheep in Europe, more recently has been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. Transmission of A. phagocytophilum is reported to be by ticks, primarily of the genus Ixodes. While mechanical and transplacental transmission of the type genus organism, A. marginale, occur in addition to tick transmission, these modes of transmission have not been considered for A. phagocytophilum. Recently, we developed a sheep model for studying host-tick-pathogen interactions of the human NY-18 A. phagocytophilum isolate. Sheep were susceptible to infection with this human isolate and served as a source of infection for I. scapularis ticks, but they did not display clinical signs of disease, and the pathogen was not apparent in stained blood smears. In the course of these experiments, one sheep unexpectedly gave birth to a lamb 5 weeks after being experimentally infected by inoculation with the pathogen propagated in HL-60 cells. The lamb was depressed and not feeding and was subsequently euthanized 18 h after birth. Tissues were collected at necropsy for microscopic examination and PCR to confirm A. phagocytophilum infection. At necropsy, the stomach contained colostrum, the spleen was moderately enlarged and thickened with conspicuous lymphoid follicles, and mesenteric lymph nodes were mildly enlarged and contained moderate infiltrates of eosinophils and neutrophils. Blood, spleen, heart, skin and cervical and mesenteric lymph nodes tested positive for A. phagocytophilum by PCR, and sequence analysis confirmed that the lamb was infected with the NY-18 isolate. Transplacental transmission should therefore be considered as a means of A. phagocytophilum transmission and may likely contribute to the epidemiology of tick-borne fever in sheep and other mammals, including humans.
Assuntos
Anaplasma phagocytophilum/isolamento & purificação , Ehrlichiose/transmissão , Placenta/microbiologia , Prenhez , Doenças dos Ovinos/transmissão , Ovinos/microbiologia , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/patogenicidade , Animais , Células Cultivadas/microbiologia , DNA Bacteriano/análise , Ehrlichiose/epidemiologia , Feminino , Humanos , Reação em Cadeia da Polimerase , Gravidez , Doenças dos Ovinos/microbiologia , Doenças Transmitidas por Carrapatos/microbiologia , Doenças Transmitidas por Carrapatos/transmissão , Doenças Transmitidas por Carrapatos/veterinária , Carrapatos/microbiologiaRESUMO
Caused mainly by Candida albicans, oropharyngeal candidiasis is the most common oral complication associated with HIV disease worldwide. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral mucosa is the first line of defense in the form of a physical barrier against C. albicans invasion, and since epithelial cells are involved in anti-Candida innate immunity through different cytokines, we wanted to determine whether C. albicans alters E-cadherin expression and production, and whether interferon-γ (INFγ), a TH1 cytokine, is involved in the anti-Candida defense. Using primary human gingival epithelial cells, we demonstrated that C. albicans significantly decreased E-cadherin mRNA expression and protein production. This effect was basically obtained at later infective periods (24 and 48h). Interestingly, when IFNγ was added to C. albicans infected epithelial cell cultures, it prevented the side effect of C. albicans on E-cadherin mRNA expression and protein production and deposition. All together, these results suggested concomitant interactions between oral epithelial cells and IFNγ against C. albicans infection.
Assuntos
Caderinas/biossíntese , Candida albicans/fisiologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Gengiva/citologia , Interferon gama/farmacologia , Western Blotting , Caderinas/genética , Adesão Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Regulação para Baixo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealAssuntos
Toxinas Bacterianas/toxicidade , Burkholderia pseudomallei/patogenicidade , Exotoxinas/fisiologia , Melioidose/microbiologia , Substituição de Aminoácidos , Animais , Antineoplásicos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas , Guerra Biológica , Burkholderia pseudomallei/genética , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/microbiologia , DNA Helicases/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Exotoxinas/genética , Exotoxinas/toxicidade , Humanos , Melioidose/diagnóstico , Melioidose/tratamento farmacológico , Terapia de Alvo Molecular , Especificidade da Espécie , Virulência/genéticaRESUMO
The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.
Assuntos
Quimiocina CXCL2/fisiologia , Haemophilus influenzae/imunologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Ligamento Espiral da Cóclea/citologia , Animais , Sítios de Ligação , Linhagem Celular/metabolismo , Linhagem Celular/microbiologia , Movimento Celular , Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Quimiocina CXCL2/biossíntese , Quimiocina CXCL2/genética , Regulação da Expressão Gênica , MAP Quinase Quinase 1/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Otite Média/imunologia , Ratos , Proteínas Recombinantes de Fusão , Transdução de Sinais , Especificidade da Espécie , Fator de Transcrição AP-1/metabolismo , Transfecção , Regulação para CimaRESUMO
In recent years galectin-3 has gained attention as a signalling molecule, mainly in inflammatory diseases. Data on galectin-3 expression in neonates, however, are limited, and expression of this lectin in cord blood has not yet been reported. The aim of this study was to determine galectin-3 levels in cord blood of term and preterm neonates as well as galectin-3 levels in cord blood of term neonates after stimulation with the prevalent pathogen Streptococcus agalactiae. Cord blood samples were incubated for 24 h and galectin-3 levels were assessed by enzyme-linked immunosorbent assay. There is a positive correlation between gestational age and galectin-3 levels in cord blood. Expression of galectin-3 is significantly higher in cord blood of small-for-gestational-age infants compared to appropriate-for-gestational-age infants. Stimulation with an invasive but not with a colonizing strain of S. agalactiae induced expression of galectin-3. Galectin-3 is expressed constitutively in cord blood of neonates and seems to play a role in the innate immunity of this population.
Assuntos
Sangue Fetal/química , Galectina 3/sangue , Recém-Nascido/sangue , Recém-Nascido Prematuro/sangue , Recém-Nascido Pequeno para a Idade Gestacional/sangue , Peso ao Nascer , Células Sanguíneas/imunologia , Células Sanguíneas/metabolismo , Células Sanguíneas/microbiologia , Células Cultivadas/imunologia , Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Etnicidade , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/imunologia , Galectina 3/biossíntese , Galectina 3/genética , Galectina 3/fisiologia , Alemanha/epidemiologia , Idade Gestacional , Humanos , Imunidade Inata , Recém-Nascido/imunologia , Recém-Nascido Prematuro/imunologia , Recém-Nascido Pequeno para a Idade Gestacional/imunologia , Masculino , Oriente Médio/etnologia , Gravidez , Complicações na Gravidez/etnologia , Complicações na Gravidez/imunologia , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/patogenicidade , Turquia/etnologiaRESUMO
Airway diseases often feature persistent neutrophilic inflammation and infection. In cystic fibrosis bronchitis, for example, Pseudomonas aeruginosa is isolated frequently. Previously, this laboratory revealed that neutrophils become major sources of histamine in mice with tracheobronchitis caused by the wall-less bacterium Mycoplasma pulmonis. To test the hypothesis that more-broadly pathogenic P. aeruginosa (which expresses cell wall-associated LPS and novel toxins) has similar effects, we incubated naïve mouse neutrophils with two strains of P. aeruginosa. Strain PAO1 greatly increased neutrophil histamine content and secretion, whereas strain PA103 depressed histamine production by killing neutrophils. The histamine-stimulating capacity of PAO1, but not PA103-mediated toxicity, persisted in heat-killed organisms. In PAO1-infected mice, lung and neutrophil histamine content increased. However, PAO1 did not alter production by mast cells (classical histamine reservoirs), which also resisted PA103 toxicity. To explore mechanisms of neutrophil-selective induction, we measured changes in mRNA encoding histidine decarboxylase (rate-limiting for histamine synthesis), probed involvement of endotoxin-TLR pathways in Myd88-deficient neutrophils, and examined contributions of pyocyanin and exotoxins. Results revealed that PAO1 increased histamine production by up-regulating histidine decarboxylase mRNA via pathways largely independent of TLR, pyocyanin, and type III secretion system exotoxins. PAO1 also increased histidine decarboxylase mRNA in neutrophils purified from infected lung. Stimulation required direct contact with neutrophils and was blocked by phagocytosis inhibitor cytochalasin D. In summary, Pseudomonas-augmented histamine production by neutrophils is strain-dependent in vitro and likely mediated by up-regulation of histidine decarboxylase. These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine.
Assuntos
Liberação de Histamina , Histamina/biossíntese , Neutrófilos/metabolismo , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/fisiologia , ADP Ribose Transferases/farmacologia , Animais , Apoptose , Toxinas Bacterianas/farmacologia , Biofilmes , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Meios de Cultivo Condicionados/farmacologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Mastócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Neutrófilos/microbiologia , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Piocianina/farmacologia , Especificidade da Espécie , Organismos Livres de Patógenos EspecíficosRESUMO
OBJECTIVE: The authors evaluated algorithms commonly used in syndromic surveillance for use as screening tools to detect potentially clonal outbreaks for review by infection control practitioners. DESIGN: Study phase 1 applied four aberrancy detection algorithms (CUSUM, EWMA, space-time scan statistic, and WSARE) to retrospective microbiologic culture data, producing a list of past candidate outbreak clusters. In phase 2, four infectious disease physicians categorized the phase 1 algorithm-identified clusters to ascertain algorithm performance. In phase 3, project members combined the algorithms to create a unified screening system and conducted a retrospective pilot evaluation. MEASUREMENTS: The study calculated recall and precision for each algorithm, and created precision-recall curves for various methods of combining the algorithms into a unified screening tool. RESULTS: Individual algorithm recall and precision ranged from 0.21 to 0.31 and from 0.053 to 0.29, respectively. Few candidate outbreak clusters were identified by more than one algorithm. The best method of combining the algorithms yielded an area under the precision-recall curve of 0.553. The phase 3 combined system detected all infection control-confirmed outbreaks during the retrospective evaluation period. LIMITATIONS: Lack of phase 2 reviewers' agreement indicates that subjective expert review was an imperfect gold standard. Less conservative filtering of culture results and alternate parameter selection for each algorithm might have improved algorithm performance. CONCLUSION: Hospital outbreak detection presents different challenges than traditional syndromic surveillance. Nevertheless, algorithms developed for syndromic surveillance have potential to form the basis of a combined system that might perform clinically useful hospital outbreak screening.
Assuntos
Algoritmos , Infecção Hospitalar/prevenção & controle , Surtos de Doenças/prevenção & controle , Controle de Infecções , Reconhecimento Automatizado de Padrão , Vigilância da População/métodos , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/prevenção & controle , Células Cultivadas/microbiologia , Infecção Hospitalar/epidemiologia , Humanos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Microbial contamination is a major issue in cell culture, but there are a range of procedures which can be adopted to prevent or eliminate contamination. Contamination may arise from the operator and the laboratory environment, from other cells used in the laboratory, and from reagents. Some infections may present a risk to laboratory workers: containment and aseptic technique are the key defence against such risks. Remedial management of suspected infection may simply mean discarding a single potentially infected culture. However, if a more widespread problem is identified, then all contaminated cultures and associated unused media that have been opened during this period should be discarded, equipment should be inspected and cleaned, cell culture operations reviewed, and isolation from other laboratories instituted until the problem is solved. Attention to training of staff, laboratory layout, appropriate use of quarantine for new cultures or cell lines, cleaning and maintenance, and quality control are important factors in preventing contamination in cell culture laboratories.