The value of antibody-coated bacteria in tracheal aspirates for the diagnosis of ventilator-associated pneumonia: a case-control study / Utilidade da avaliação de bactérias revestidas por anticorpos em aspirados traqueais para o diagnóstico de pneumonia associada à ventilação mecânica: um estudo caso-controle

ABSTRACT Objective: Ventilator-associated pneumonia (VAP) is the leading type of hospital-acquired infection in ICU patients. The diagnosis of VAP is challenging, mostly due to limitations of the diagnostic methods available. The aim of this study was to determine whether antibody-coated bacteria (ACB) evaluation can improve the specificity of endotracheal aspirate (EA) culture in VAP diagnosis. Methods: We conducted a diagnostic case-control study, enrolling 45 patients undergoing mechanical ventilation. Samples of EA were obtained from patients with and without VAP (cases and controls, respectively), and we assessed the number of bacteria coated with FITC-conjugated monoclonal antibodies (IgA, IgM, or IgG) or an FITC-conjugated polyvalent antibody. Using immunofluorescence microscopy, we determined the proportion of ACB among a fixed number of 80 bacteria. Results: The median proportions of ACB were significantly higher among the cases (n = 22) than among the controls (n = 23)-IgA (60.6% vs. 22.5%), IgM (42.5% vs. 12.5%), IgG (50.6% vs. 17.5%), and polyvalent (75.6% vs. 33.8%)-p < 0.001 for all. The accuracy of the best cut-off points for VAP diagnosis regarding monoclonal and polyvalent ACBs was greater than 95.0% and 93.3%, respectively. Conclusions: The numbers of ACB in EA samples were higher among cases than among controls. Our findings indicate that evaluating ACB in EA is a promising tool to improve the specificity of VAP diagnosis. The technique could be cost-effective and therefore useful in low-resource settings, with the advantages of minimizing false-positive results and avoiding overtreatment.

RESUMO Objetivo: A pneumonia associada à ventilação mecânica (PAVM) é o principal tipo de infecção adquirida no ambiente hospitalar em pacientes em UTIs. O diagnóstico de PAVM é desafiador, principalmente devido a limitações dos métodos diagnósticos disponíveis. O objetivo deste estudo foi determinar se a avaliação de bactérias revestidas por anticorpos (BRA) pode melhorar a especificidade de culturas de aspirado traqueal (AT) no diagnóstico de PAVM. Métodos: Estudo diagnóstico caso-controle envolvendo 45 pacientes sob ventilação mecânica. Amostras de AT foram obtidas de pacientes com e sem PAVM (casos e controles, respectivamente), e verificamos o número de bactérias revestidas com anticorpos monoclonais conjugados com FITC (IgA, IgM ou IgG) ou anticorpo polivalente conjugado com FITC. Utilizando microscopia de imunofluorescência, foi determinada a proporção de BRA em um número fixo de 80 bactérias. Resultados: A mediana das proporções de BRA foi significativamente maior nos casos (n = 22) que nos controles (n = 23) - IgA (60,6% vs. 22,5%), IgM (42,5% vs. 12,5%), IgG (50,6% vs. 17,5%) e polivalente (75,6% vs. 33,8%) - p < 0,001 para todos. A acurácia dos melhores pontos de corte para o diagnostico de PAVM em relação aos BRA monoclonais e polivalentes foi > 95,0% e > 93,3%, respectivamente. Conclusões: O número de BRA em amostras de AT foi maior nos casos que nos controles. Nossos achados indicam que a avaliação de BRA no AT é uma ferramenta promissora para aumentar a especificidade do diagnóstico de PAVM. A técnica pode ser custo-efetiva e, portanto, útil em locais com poucos recursos, com as vantagens de minimizar resultados falso-positivos e evitar o tratamento excessivo.

Hiperinsuflação pulmonar com ventilador mecânico versus aspiração traqueal isolada na higiene brônquica de pacientes submetidos à ventilação mecânica / Lung hyperinflation by mechanical ventilation versus isolated tracheal aspiration in the bronchial hygiene of patients undergoing mechanical ventilation

RESUMO Objetivo: Determinar a eficácia da manobra de hiperinsuflação pulmonar com o ventilador mecânico, em comparação à aspiração traqueal isolada, para remover secreções, normalizar a hemodinâmica e melhorar a mecânica pulmonar em pacientes em ventilação mecânica. Métodos: Ensaio clínico randomizado cruzado incluindo pacientes em ventilação mecânica por mais de 48 horas internados na unidade de terapia intensiva. Os pacientes foram randomizados para receber a aspiração traqueal isolada (Grupo Controle) e hiperinsuflação pulmonar por meio do ventilador mecânico (Grupo HVM). Mensuraram-se parâmetros hemodinâmicos e de mecânica respiratória, assim como a quantidade de secreção aspirada. Resultados: Foram incluídos 50 pacientes. A média de idade dos pacientes foi de 44,7 ± 21,6 anos, sendo 31 do sexo masculino. O Grupo HVM apresentou maior quantidade de secreção aspirada (3,9g versus 6,4g; p = 0,0001), variação na média da complacência dinâmica (-1,3 ± 2,3 versus -2,9 ± 2,3; p = 0,008), volume corrente expirado (-0,7 ± 0,0 versus -54,1 ± 38,8; p = 0,0001) e diminuição significativa da pressão inspiratória de pico (0,2 ± 0,1 versus 2,5 ± 0,1; p = 0,001), em comparação com o Grupo Controle. Conclusão: Na amostra estudada, a técnica de HVM apresentou maior quantidade de secreção aspirada, aumento significativo da complacência dinâmica e volume corrente expirado, além de diminuição significativa da pressão de pico inspiratória.

ABSTRACT Objective: To determine the efficacy of lung hyperinflation maneuvers via a mechanical ventilator compared to isolated tracheal aspiration for removing secretions, normalizing hemodynamics and improving lung mechanics in patients on mechanical ventilation. Methods: This was a randomized crossover clinical trial including patients admitted to the intensive care unit and on mechanical ventilation for more than 48 hours. Patients were randomized to receive either isolated tracheal aspiration (Control Group) or lung hyperinflation by mechanical ventilator (MVH Group). Hemodynamic and mechanical respiratory parameters were measured along with the amount of aspirated secretions. Results: A total of 50 patients were included. The mean age of the patients was 44.7 ± 21.6 years, and 31 were male. Compared to the Control Group, the MVH Group showed greater aspirated secretion amount (3.9g versus 6.4g, p = 0.0001), variation in mean dynamic compliance (-1.3 ± 2.3 versus -2.9 ± 2.3; p = 0.008), and expired tidal volume (-0.7 ± 0.0 versus -54.1 ± 38.8, p = 0.0001) as well as a significant decrease in peak inspiratory pressure (0.2 ± 0.1 versus 2.5 ± 0.1; p = 0.001). Conclusion: In the studied sample, the MVH technique led to a greater amount of aspirated secretions, significant increases in dynamic compliance and expired tidal volume and a significant reduction in peak inspiratory pressure.

Transtracheal puncture: a forgotten procedure

Transtracheal puncture has long been known as a safe, low-cost procedure. However, with the advent of bronchoscopy, it has largely been forgotten. Two researchers have suggested the use of α-amylase activity to diagnose salivary aspiration, but the normal values of this enzyme in tracheobronchial secretions are unknown. We aimed to define the normal values of α-amylase activity in tracheobronchial secretions and verify the rate of major complications of transtracheal puncture. From October 2009 to June 2011, we prospectively evaluated 118 patients without clinical or radiological signs of salivary aspiration who underwent transtracheal puncture before bronchoscopy. The patients were sedated with a solution of lidocaine and diazepam until they reached a Ramsay sedation score of 2 or 3. We then cleaned the cervical region and anesthetized the superficial planes with lidocaine. Next, we injected 10 mL of 2% lidocaine into the tracheobronchial tree. Finally, we injected 10 mL of normal saline into the tracheobronchial tree and immediately aspirated the saline with maximum vacuum pressure to collect samples for measurement of the α-amylase level. The α-amylase level mean ± SE, median, and range were 1914 ± 240, 1056, and 24-10,000 IU/L, respectively. No major complications (peripheral desaturation, subcutaneous emphysema, cardiac arrhythmia, or hemoptysis) occurred among 118 patients who underwent this procedure. Transtracheal aspiration is a safe, low-cost procedure. We herein define for the first time the normal α-amylase levels in the tracheobronchial secretions of humans.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Post-mortem re-cloning of a transgenic red fluorescent protein dog

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

beta-catenin/Tcf signaling in squamous differentiation of porcine airway epithelial cells / 华中科技大学学报（医学）（英德文版）

For a preliminary study of the role of beta-catenin/Tcf signaling in squamous differentiation of airway (tracheobronchial) epithelial cells, a stable mutant of beta-catenin was transfected into primarily cultured porcine airway epithelial cells. Western blotting revealed that exogenous protein was observed in large quantity in cytoplasm and nucleus. When co-transfected with Tcf luciferase reporter plasmids, beta-catenin mutant increased the reporter's transcriptional activities. However, mRNA expression of a squamous differentiation marker, small proline-rich protein (SPRP), was not elevated, as shown by reverse transcription-polymerase chain reaction. These findings suggest that beta-catenin/Tcf signaling may not be directly involved in the squamous differentiation of porcine airway epithelial cells.

Change of connexin 37 in allergen-induced airway inflammation

Gap junction channels formed with connexins directly link to the cytoplasm of adjacent cells and have been implicated in intercellular signaling. Connexin 37 (Cx37) is expressed in the gas-exchange region of the lung. Recently, Cx37 has been reported to be involved in the pathogenesis of inflammatory disease. However, no data are available on the role of Cx37 in allergic airway inflammatory disease. In the present study, we used a murine model of ovalbumin (OVA)-induced allergic airway disease and primary murine epithelial cells to examine the change of Cx37 in allergic airway disease. These mice develop the following typical pathophysiological features of asthma: airway hyperresponsiveness, airway inflammation, and increased IL-4, IL-5, IL-13, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, eotaxin, and RANTES levels in lungs. Cx37 protein and mRNA expression were decreased in OVA-induced allergic airway disease. Immunoreactive Cx37 localized in epithelial layers around the bronchioles in control mice, which dramatically disappeared in allergen-induced asthmatic lungs. Moreover, the levels of Cx37 protein in lung tissues showed significantly negative correlations with airway inflammation, airway responsiveness, and levels of Th2 cytokines in lungs. These findings indicate that change of Cx37 may be associated with the asthma phenotype.

The expression and significance of IgE in anaphylactic shock guinea-pigs / 法医学杂志

OBJECTIVE@#To seek the pathomorphological targets for forensic expertise in anaphylactic shock.@*METHODS@#The expression of IgE in hearts, lungs, livers, spleens, kidneys, gastrics, intestinals, tracheas and tonsils of anaphylactic shock guinea-pigs was observed at 0, 6, 12 h and 24 h respectively by tissue chip S-P immuno-histochemical method.@*RESULTS@#Positive expression of IgE presented in lungs and tracheas in the test group with the peak at 0 hour and it declined as time advanced, and also there were significant differences at different times (P<0.05).@*CONCLUSION@#The immuno-histochemical method of detecting the expression of IgE in lungs, tracheas and spleens can be supposed to be the pathomorphological targets for forensic expertise in anaphylactic shock. The weakening of the positive expression of IgE in lungs and tracheas as the time advanced suggested that in this kind of case the autopsy should be arried out as early as possible.

Clinical Implications of the Glucose Test Strip Method for Early Detection of Pulmonary Aspiration in Nasogastric Tube- Fed Patients / 간호학회지

PURPOSE: This study was performed to test the clinical usefulness of the glucose test strip method for early detection of pulmonary aspiration in tube fed patients. METHOD: The subjects for the study were 36 patients who were receiving enteral feedings and 39 patients who were not given enteral feedings. For the analysis, the tube fed patients were divided into two groups (clinically significant aspiration and no aspiration) according to criteria. RESULT: The mean glucose concentration of tracheal secretions from non enteral fed patients was 26.35mg/dl and were lower than those concentrations found in tube fed patients (32.75mg/dl). The mean glucose concentration of the aspiration group was 45.60mg/dl and the glucose concentration of the non aspiration group was 19.93mg/dl. The difference was statistically significant (t=2.163, p=. 038). More subjects in the no aspiration group (73%) than the aspiration group (56%) had glucose concentrations below 20mg/dl. After deleting the cases that had samples containing blood, glucose concentrations of tracheal aspirates were lower in both groups. CONCLUSION: The glucose level of the aspiration group was significantly lower than the no aspiration group and more subjects in the aspiration group had a glucose level higher than 101mg/dl. Therefore, the glucose test of tracheal secretions in tube fed patients could be a desirable test for screening for tracheal aspiration. Especially the patient who is showing repeatedly high glucose levels should not be given feedings until reassessment is completed.

Upregulation of the tracheobronchial mucin gene involves cyclic AMP response elements.

The cAMP response element (CRE)-binding transcription factor CREB can mediate induction of gene transcription in response to cAMP. Since the tracheobronchial mucin gene (TBM) 5'-flanking region contains CREs (located between residues -289 and -376) with an octamer-like motif (TGACGTCC), the cAMP responsiveness of the TBM CREs was investigated in human tracheal epithelial cells HBE1. These cells were isolated from non-cystic fibrosis subjects and immortalized with HPV18 genes E6 and E7 (ref. 1). HBE1 cells express a homolog of canine TBM (as demonstrated by TBM expression at the transcription and translation level). Electrophoretic mobility shift assay (EMSA) indicated that CREs provide a binding site for nuclear proteins. Transient transfection analysis [using the chloramphenicol acetyl transferase (CAT) reporter gene] and nuclear run on analysis indicated cAMP induced transcription of the TBM gene. The transcriptional activity of the HBE1 transfected cells containing CRE was selectively modulated by extracellular 8Br-cAMP in a dose-dependent manner; a 6-fold increase in activity was detected when cells were incubated for 12 hr in the presence of 2 microM vs 1 nM 8BrcAMP. Since mucin gene is over-expressed in diseases such as cystic fibrosis (CF) and asthma, the information presented here will help us understand the mechanisms involved in transcriptional regulation of mucin gene expression in disease states.

Subcellular distribution and pharmacological characterization of muscarinic receptors in tracheal smooth muscle

: Subcellular fractions isolated from tracheal smooth muscle have been identified using biochemical markers and measuring the [3H]QNB muscarinic receptor binding activity in these fractions. This muscarinic receptor (mAchR) activity was slightly enriched 1.6 times in the crude mitochondrial fraction (M), 2.6 times in the crude microsomal fraction (P), and greatly enriched in the highly purified plasma membranes fractions, being 5.3 times in a heavy plasma membrane fraction designed as P2 and 9.1 times in a light plasma membrane fraction named P1 fraction. The muscarinic receptor subtypes present in the subcellular fractions were identified using competition experiments. The binding of five selective antagonists, pirenzepine, AF-DX 116, hexahydrodifenidol, methoctramine and 4-DAMP were examined. In this sense, the M1 antagonist pirenzepine showed pKi's values between 6.44-7.45 and the M2 antagonist AF-DX 116 showed pKi's values ranging from 6.75 to 7.45 being the lowest pKi's values here described. The antagonist hexahydrodifenidol showed higher affinities than pirenzepine-derivated compounds with pKi's values from 7.25 to 7.65. The antagonist 4-DAMP exhibited pKi's values from 8.18-8.41. Finally, methoctramine showed similar affinities as 4-DAMP, with pKi's ranging from 8.09 to 8.22 suggesting the existence of M2 receptors in these fractions. These data suggest that M2 mAchR are present in all articulate fractions here studied. It is important to emphasize that the M2 muscarinic receptor presents in the light plasma membrane fraction (P1) shows poor selectivity towards the muscarinic antagonists being different from the M2 mAchRs associated with other subcellular fractions isolated from bovine tracheal smooth muscle

Novo sistema de umidificaçäo e aquecimento da mistura gasosa dos respiradores: estudo comparativo / The new system of gaseous mixture humidification and heating of respirators: comparative study

Foram estudados dois grupos de pacientes comparando-se o desempenho de nebulizaçäo convencional em relaçäo a um novo sistema constituído pela interposiçäo de um condensador higroscópico no circuito sem nebulizador. A eficiência da umidificaçäo foi relativamente igual nos dois grupos, sendo que os pacientes com nebulizaçäo convencional tiveram estatísticamente maior formaçäo de "rolhas". A contaminaçäo, apesar de trocas diárias dos circuitos, e de 8// com o uso dos condensadores, mesmo em circuitos que permanecram sem troca por até nove dias. Esta diferença foi altamente signficativa (p = 0,0011). O preço comparativo dos dois sistemas mostrou que o custo do uso do condensador é de menos da metade do custo da troca e desinfecçäo diária de circuitos (221,64 doólares contra 537,30 dólares por mês do uso contínuo do respirador)

Peptide mapping reveals differences in the non-glycosylated domains of cystic fibrosis and normal tracheobronchial mucins.

Tracheobronchial mucins from lung mucus secretions of healthy individuals and from patients with cystic fibrosis (CF) were purified according to a protocol established in our laboratory. Following digestion of the purified, reduced-alkylated mucin (free of 118 kDa and 70 kDa components) with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2 and TR-3) were observed upon chromatography on a Superose 6 column using FPLC. TR-1 (glycosylated fraction) contained all of the carbohydrate, while TR-2 and TR-3 fractions had no detectable sugars. Comparison of the amino acid composition of TR-1 fractions from normal and CF individuals revealed no significant differences, while the TR-2 fractions from these mucins showed noticeable differences. Peptide mapping of TR-2 fractions from normal and CF mucins was performed on a C18 reverse phase column using FPLC. The peptide maps of normal mucins were markedly different from CF mucins. A greater number of peptides were seen in the TR-2 fractions of normal mucins when compared to CF mucin TR-2 fractions. In addition, normal TR-2 fractions appeared to be comprised of more hydrophobic peptides when compared to CF TR-2 fractions. These data provide evidence of possible structural differences in the non-glycosylated regions of CF and non-CF mucins, since the TR-2 fractions are essentially derived from the T-domains in the "naked" stretches of the mucin polypeptide backbone.

Biochemical and pharmacological characterization of octylglucoside-solubilized muscarinic receptors derived from bovine tracheal smooth muscle

La actividad de receptor colinérgico muscarínico fue investigada en las fracciones subcelulares del músculo liso traqueal de bovino. Esta actividad de receptor muscarínico fue determinada mediante un ensayo basado en la unión específica del H-QNB (antagonista muscarínico Bencilato de Quinuclidinilo tritiado) utilizando un método simple y reproducible que permite separar el ligando radioactivo que se encuentra libre del unido, mediante un procedimiento de centrifugación en columnas; lo cual permite obtener valores muy bajos como "blanco". Esta actividad de receptor muscarínico fue encontrada enriquecida 4 veces en la fracción microsomal (P) con respecto al Extracto original y después de una etapa adicional de purificación utilizando un gradiente discontinuo de sacarosa se concentró 7 veces dicha actividad en la fracción de membranas plasmáticas (P-1). A partir de esta fracción de membranas plasmáticas enriquecida en receptor muscarínico y utilizando un método original que emplea un detergente no iónico como es el Octilglucósido al 1% se obtuvo una fracción de receptor muscarínico "solubilizado", después de centrifugar a 200.000 x g x 30 min. El material solubilizado fue filtrado a través de una columna de Sephadex G-50, con la finalidad de remover el detergente y el eluente obtenido de esta columna se denominó receptor muscarínico "solubilizado". El procedimiento descrito inicialmente para evaluar la interacción entre el receptor muscarínico y el H-QNB, también permite determinar bajo las mismas condiciones experimentales, la unión del H-QNB, tanto a los receptores muscarínicos unidos a las membranas plasmáticas como a los receptores presentes en el "solubilizado". Se encontró que el receptor muscarínico "solubilizado" y el asociado a las membranas plasmáticas mostraron idénticas propiedades bioquímicas y farmacológicas