Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities

Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

Evaluation of potential biocontrol agents against Claviceps africana in vitro and in vivo

Undiluted culture filtrates of two commercial products of Trichoderma spp., Trichopel and Trichoflow, and two isolates of Penicillium citrinum completely inhibited the conidial germination of macroconidia of Claviceps africana , the cause of ergot or sugary disease of sorghum (Sorghum bicolor) in vitro . Similarly, Pseudomonas aeruginosa and Burkholderia cepacia completely inhibited macroconidial germination, with the former being more effective at high dilutions. In contrast, these bacterial isolates failed to inhibit infection in vivo in glasshouse tests with ergot-inoculated sorghum, but all fungal biocontrol agents (including an isolate of Epicoccum nigrum) reduced the severity of disease (percentage of infected spikelets per panicle), in some cases completely inhibiting the development of ergot. In a second glasshouse trial, optimum control was achieved when the biocontrol agents were applied 3-7 days before inoculation with conidia of C. africana .

The genetics of glycosylation in Gram-negative bacteria

In recent years there has been a dramatic increase in reports of glycosylation of proteins in various Gram-negative systems including Neisseria meningitidis, Neisseria gonorrhoeae, Campylobacter jejuni, Pseudomonas aeruginosa, Escherichia coli, Caulobacter crescentus, Aeromonas caviae and Helicobacter pylori. Although this growing list contains many important pathogens (reviewed by Benz and Schmidt [Mol. Microbiol. 45 (2002) 267-276]) and the glycosylations are found on proteins important in pathogenesis such as pili, adhesins and flagella the precise role(s) of the glycosylation of these proteins remains to be determined. Furthermore, the details of the glycosylation biosynthetic process have not been determined in any of these systems. The definition of the precise role of glycosylation and the mechanism of biosynthesis will be facilitated by a detailed understanding of the genes involved. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Pericondrite por piercing: relato de casos e revis��o da literatura

A pericondrite �� uma infec����o que envolve o peric��ndrio do pavilh��o auricular. Desencadeada pelo piercing, �� secund��ria a uma rea����o al��rgica ao n��quel. O diagn��stico �� essencialmente cl��nico, por��m a realiza����o de cultura e antibiograma �� fundamental. Os pat��genos freq��entemente envolvidos s��o germes Gram negativos (Pseudomonas aeruginosa). Os autores apresentam 3 casos de pericondrite por piercing atendidos no ambulat��rio de otorrinolaringologia e realizam uma revis��o da literatura.

Bacteriologia do aspirado do meato m��dio em pacientes com fibrose c��stica

A combina����o de fatores como viscosidade das secre����es dos seios paranasais, diminui����o da drenagem sinusal e comprometimento do transporte mucociliar podem ser respons��veis pela cria����o de um ambiente prop��cio e adequado para a coloniza����o de bact��rias nos seios paranasais de pacientes com fibrose c��stica. OBJETIVO: Analisar a bacteriologia do aspirado do meato m��dio de pacientes portadores de fibrose c��stica. MATERIAL E M��TODO: Atrav��s de um estudo prospectivo de delineamento transversal, avaliou-se uma amostra composta de 23 pacientes, avaliados durante 2 anos. Realizaram-se rela����es entre a cultura do meato m��dio e a avalia����o radiol��gica do seio maxilar e a avalia����o cl��nica. Secundariamente, estudou-se a rela����o da bacteriologia do aspirado do meato m��dio e a do escarro. RESULTADOS: No total foram realizadas 42 aspira����es do meato m��dio. Em 17 (73,91%) dos 23 pacientes, as culturas foram negativas e, em 6 (26,08%), positivas. Das 42 aspira����es, 31 (73,8%) foram negativas e 11 (26,2%), positivas. A presen��a de Pseudomonas aeruginosa foi observada em 18,18% das culturas positivas e o Staphylococcus aerus em 27,28%. CONCLUS��O: A maioria das culturas do aspirado do meato m��dio de pacientes com fibrose c��stica foi negativa.

Atividade antimicrobiana in vitro de produtos vegetais em otite externa aguda

Otite externa aguda �� a inflama����o do conduto auditivo externo, e plantas medicinais podem ser utilizadas, na cultura popular, para seu tratamento. OBJETIVO: Avaliar atividade antimicrobiana in vitro de Aleolanthus suaveolens, Caryophyllus aromaticus, Cymbopogon citratus, Matricaria chamomila, Pithecellobium avaremotemo, Plectranthus amboinicus e Ruta graveolens sobre agentes etiol��gicos de otite externa. CASU��STICA E M��TODOS: A concentra����o inibit��ria m��nima de extratos e ��leos destas plantas foi obtida em amostras de otite externa. RESULTADOS: Staphylococcus aureus em 10 culturas, Pseudomonas aeruginosa em 8, Pseudomonas aeruginosa e Staphylococcus aureus, em associa����o, em 5 culturas e Candida albicans e Candida krusei em 4 culturas. P. aeruginosa foi resistente a todos os extratos e ��leos essenciais testados; os extratos de A. suaveolens, P. avaremotemo e de R. graveolens foram inativos, o ��leo essencial de C. aromaticus e M. chamomila foram ativos contra 3 cepas de S. aureus e as cepas de Candida; Sete das cepas de S. aureus foram sens��veis ao extrato de P. amboinicus, mas o ��leo n��o mostrou atividade, 4 cepas de S.aureus e as cepas de Candida foram sens��veis ao ��leo essencial de R. graveolens. CONCLUS��O: Algumas plantas apresentaram resultados satisfat��rios, dependendo do agente etiol��gico, por��m se faz necess��rio estudos mais detalhados, para melhorar o aproveitamento destas plantas.

Pericondrite p��s-piercing

O uso de piercing tem se tornado uma pr��tica muito freq��ente entre os jovens. O procedimento na maioria vezes realizado por profissionais n��o-qualificados n��o �� isento de riscos. O manuseio de material contaminado ou a higiene impr��pria predisp��em �� pericondrite e �� celulite. A pericondrite caracteriza-se pelo eritema do pavilh��o auricular, dor intensa e febre. Sem tratamento, desenvolve-se um edema generalizado do pavilh��o com forma����o de abscesso subpericondrial, podendo evoluir para necrose isqu��mica da cartilagem e a tem��vel deformidade est��tica conhecida como "orelha em couve flor". O agente respons��vel mais encontrado �� o Pseudomonas aeruginosa. No est��gio inicial da doen��a o tratamento pode ser feito com antibi��ticos de amplo espectro. Nos casos em que o abscesso est�� presente, a incis��o e drenagem cir��rgica s��o obrigat��rios acompanhado de antibioticoterapia guiado pela cultura e antibiograma. OBJETIVO: O objetivo deste relato de caso �� realizar uma revis��o bibliogr��fica dos ��ltimos 10 anos abordando os aspectos anat��micos do pavilh��o auricular, a hist��ria do uso de piercing e suas mais conhecidas complica����es. M��TODO: Relato de um caso de pericondrite p��s-piercing transcartilaginoso onde houve a necessidade de tratamento cir��rgico com praticamente nenhuma deformidade est��tica. RESULTADO: Aquisi����o de experi��ncia te��rico-pr��tica atrav��s de revis��o bibliogr��fica e relato de um caso de evolu����o favor��vel para a paciente. CONCLUS��O: Incid��ncia crescente das complica����es de pericondrites na popula����o jovem deve levar �� preven����o prim��ria mais elaborada.

Atividade antimicrobiana, antioxidante e teor de compostos fen��licos em casca e am��ndoa de frutos de manga

O processamento industrial de frutos de manga gera elevada quantidade de res��duos agroindustriais, representados pelas cascas e sementes (am��ndoa), os quais, sem aplica����o vi��vel, acabam sendo descartados diretamente no meio ambiente. Esses res��duos s��o ricos em compostos bioativos, amplamente reconhecidos pelas suas propriedades promotoras da sa��de e em aplica����es tecnol��gicas. Os objetivos foram avaliar as atividades antimicrobiana e antioxidante e o teor de compostos fen��licos de dois extratos: um obtido da casca (FC) e outro da am��ndoa (FA) de manga variedade 'Tommy Atkins'. O teor de compostos fen��licos totais variou de 3.123 a 6.644 mg de catequina/100 g. Os extratos FC e FA demonstraram relevante atividade antimicrobiana frente ��s cepas das bact��rias Escherichia coli, Salmonella sp., Pseudomonas aeruginosa . Staphylococcus aureus, determinadas pelo m��todo de difus��o em disco. A a����o antioxidante de FC e FA aumentou com o aumento das concentra����es testadas, atingindo o valor m��ximo de 88% (FC). Em todas as concentra����es testadas os extratos FC apresentaram a����o antioxidante significativamente superior ��s respectivas concentra����es dos extratos FA. Esses resultados sugerem potencial aplica����o dos res��duos de manga como fonte de compostos fen��licos, subst��ncias antimicrobianas e antioxidantes, podendo ser explorados pelas ind��strias de alimentos.

Antibacterial activity of Baccharis trimera (Less.) DC. (carqueja) against bacteria of medical interest

Baccharis trimera (Less.) (Asteraceae), popularly know as "carqueja", is a species commonly used in folk medicine for the treatment or prevention of diseases. In this context, the purpose of this work was to study the antibacterial activity of crude hydroalcoholic extract from Baccharis trimera against Gram-positive bacterial strains (Staphylococcus aureus ATCC 29213, Staphylococcus saprophyticus ATCC 15305, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 19433) and Gram-negative bacteria (Escherichia coli EHEC ATCC 43895, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 27736, Salmonella typhi ATCC 19430) of clinical interest. Antibacterial susceptibility was evaluated by broth microdilution assay following the CLSI (formerly the NCCLS) guidelines. The extract from B. trimera showed antibacterial activity against Gram-positive bacteria and the most interesting result was obtained against S. epidermidis that presented Minimal Inhibitory Concentration of 250μg/mL. These results indicate that B. trimera have bacterisostatic potential against Gram-positive bacterial strains of medical interest and could serve as a base for further studies on the use of isolated compounds from this species as future antimicrobials.

Coeficiente fen��lico na avalia����o microbiol��gica de desinfetantes de uso hospitalar e dom��stico

Analisou-se o coeficiente fen��lico de 24 desinfetantes comercializados em S��o Paulo (Brasil). Seis produtos eram de uso hospitalar e os restantes de uso dom��stico. Os compostos ativos eram �� base de fen��is, am��nio quatern��rio, formalde��do, etanol e cloro, sendo que alguns estavam associados. Os microrganismos utilizados foram Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442 e Salmonella choleraesuis ATCC 10708. Os valores dos coeficientes fen��licos variaram de 58,3 a 0,1. Os desinfetantes hospitalares mostraram valores superiores aos de uso dom��stico, mas estas diferen��as, proporcionalmente, n��o significaram melhor qualidade dos mesmos. O m��todo microbiol��gico adotado mostrou que alguns produtos, de uso dom��stico, aparentemente possuiam atividade antibacteriana baixa ou inexistente porque o coeficiente fen��lico n��o p��de ser obtido nas dilui����es utilizadas na avalia����o.

Avalia����o microbiol��gica de desinfetantes qu��micos de uso dom��stico

Em 1988 e no primeiro semestre de 1989 cinco desinfetantes de uso dom��stico foram divulgados atrav��s de publica����o televisiva. Para avaliar as propriedades antimicrobianas desses produtos os mesmos foram testados por um m��todo qualitativo (Dilui����o-Uso com 10 carreadores, m��todo convencional e outro simplificado adaptado) e outro quantitativo, tamb��m adaptado. Os compostos ativos dos produtos descritos nos respectivos r��tulos foram: 1- Paraclorofenol (O- Benzil) 0,1%; 2 - ��ter 2,4,4' Cloro (III) 2' hidroxifenilico 0,1%; 3 - N-alquildimetil benzil am��nio-Cloreto de alquil dimetil etil benzil amonio 50% - 1,6%; 4 - formalde��do 37% (solu����o de 0,3%); 5 - Sem informa����o. Os microrganismos utilizados foram: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442 e Salmonella choleraesuis ATCC 10708. No m��todo qualitativo, a cepa de pseudomonas foi recuperada dos desinfetantes 1, 2 e 3. Todos os desinfetantes mostraram um efeito germicida de 5,0 (99,999% de redu����o) em 15 segundos frente ��s tr��s cepas. O desinfetante 3 estava contaminado com Enterobacter sp na ordem de 10(4) c��lulas por ml. Este contaminante foi sens��vel diante dos desinfetantes 1, 4 e 5, frente �� metodologia qualitativa, e relativamente resistente frente ao desinfetante 2, na metodologia quantitativa.

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonasaeruginosa on immobilized metal chelates

The aim of this work was to devise a one-step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild-type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)-IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 +/- 0.015 and 3.214 +/- 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K(D) of 4.53 x 10(-7) M was obtained from batch isotherm measurements. The combination of tailor-made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one-step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial-Zn(II) and EPI-30-IDA-Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A-Sepharose CL-4B. This MAb preparation revealed on SDS-PAGE two protein bands with M(r) of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright (C) 2011 John Wiley & Sons, Ltd.