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1.
Ultrasound Obstet Gynecol ; 54(6): 759-766, 2019 Dec.
Article in English | MEDLINE | ID: mdl-30834623

ABSTRACT

OBJECTIVES: The congenital lung malformation volume ratio (CVR) is a prenatal ultrasound measurement that parameterizes congenital lung malformation (CLM) size. The aims of this study were to use serial measurements to create estimated growth curves of fetal CVR for asymptomatic and symptomatic neonates with CLM and to investigate whether a discriminant prognostic model based on these measurements could predict accurately which fetuses with CLM will require invasive respiratory support at delivery and should therefore be delivered at a tertiary-care facility. METHODS: This was a retrospective study of fetuses diagnosed prenatally with CLM at three tertiary-care children's hospitals between 2009 and 2016. Those with two or more sonographic measurements of CVR were included. Serial fetal CVR measurements were used to create estimated growth curves for neonates with and those without respiratory symptoms at delivery, defined as requiring invasive respiratory support for the first 24 h after delivery. A discriminant model based on serial CVR measurements was used to calculate the dynamic probability of the need for invasive respiratory support. The performance of this model overall and in preterm and term neonates was compared with those using maximum CVR thresholds of 1.0 and 1.6. RESULTS: Of the 147 neonates meeting the inclusion criteria, 16 (10.9%) required postnatal invasive respiratory support. The estimated CVR growth curve models showed different growth trajectories for asymptomatic and symptomatic neonates, with significantly higher CVR in symptomatic neonates, and values peaking late in the second trimester at around 25 weeks' gestation in asymptomatic neonates. All prognostic methods had high accuracy for the prediction of the need for invasive respiratory support in term neonates, but the discriminant model had the best performance overall (area under the receiver-operating characteristics curve (AUC) = 0.88) and in the preterm population (AUC = 0.85). CONCLUSIONS: The estimated CVR growth curves showed different growth patterns in asymptomatic and symptomatic neonates with CLM. The dynamic discriminant model performed well overall and particularly in neonates that were carried to term. Development of an externally validated clinical tool based on this analysis could be useful in determining the site of delivery for fetuses with CLM. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.


Subject(s)
Lung Diseases/congenital , Lung Diseases/diagnostic imaging , Lung/abnormalities , Respiration, Artificial/statistics & numerical data , Respiratory Distress Syndrome, Newborn/diagnostic imaging , Ultrasonography, Prenatal/methods , Female , Fetus , Gestational Age , Growth Charts , Humans , Infant, Newborn , Lung/diagnostic imaging , Lung/pathology , Lung Diseases/pathology , Lung Volume Measurements/methods , Predictive Value of Tests , Pregnancy , Pregnancy Trimester, Second , Prenatal Care/standards , Prognosis , Respiration, Artificial/trends , Respiratory Distress Syndrome, Newborn/etiology , Respiratory Distress Syndrome, Newborn/therapy , Retrospective Studies
2.
Mol Immunol ; 66(2): 139-46, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25771180

ABSTRACT

The development of nanoparticles (NPs) for commercial products is undergoing a dramatic expansion. Many sunscreens and cosmetics now use zinc oxide (ZnO) or titania (TiO2) NPs, which are effective ultraviolet (UV) filters. Zinc oxide topical creams are also used in mild anti-inflammatory treatments. In this study we evaluated the effect of size and dispersion state of ZnO and TiO2 NPs, compared to "bulk" ZnO, on mast cell degranulation and viability. ZnO and TiO2 NPs were characterized using dynamic light scattering and disc centrifugation. Rat basophilic leukaemia (RBL-2H3) cells and primary mouse bone marrow-derived mast cells (BMMCs) were exposed to ZnO and TiO2 NPs of different sizes (25-200 nm) and surface coatings at concentrations from 1 to 200 µg/mL. The effect of NPs on immunoglobulin E (IgE)-dependent mast cell degranulation was assessed by measuring release of both ß-hexosaminidase and histamine via colorimetric and ELISA assays. The intracellular level of Zn(2+) and Ca(2+) ions were measured using zinquin ethyl ester and Fluo-4 AM fluorescence probes, respectively. Cellular viability was determined using the soluble tetrazolium-based MTS colorimetric assay. Exposure of RBL-2H3 and primary mouse BMMC to ZnO NPs markedly inhibited both histamine and ß-hexosaminidase release. This effect was both particle size and dispersion dependent. In contrast, TiO2 NPs did not inhibit the allergic response. These effects were independent of cytotoxicity, which was observed only at high concentrations of ZnO NPs, and was not observed for TiO2 NPs. The inhibitory effects of ZnO NPs on mast cells were inversely proportional to particle size and dispersion status, and thus these NPs may have greater potential than "bulk" zinc in the inhibition of allergic responses.


Subject(s)
Basophils/drug effects , Mast Cells/drug effects , Nanoparticles/chemistry , Zinc Oxide/pharmacology , Animals , Basophils/cytology , Basophils/immunology , Calcium/metabolism , Cations, Divalent , Cell Degranulation/drug effects , Cell Degranulation/immunology , Cell Line, Tumor , Cell Survival/drug effects , Histamine/metabolism , Histamine Release/drug effects , Mast Cells/cytology , Mast Cells/immunology , Mice , Mice, Inbred C57BL , Nanoparticles/ultrastructure , Particle Size , Primary Cell Culture , Rats , Titanium/pharmacology , Zinc/metabolism , beta-N-Acetylhexosaminidases/metabolism
3.
Biosens Bioelectron ; 23(7): 1131-6, 2008 Feb 28.
Article in English | MEDLINE | ID: mdl-18155516

ABSTRACT

There is an ongoing need for field-deployable biosensor devices. We have constructed a fully self-contained, hand-held biosensor, based on the surface plasmon resonance technique. The dimensions of the sensor unit are 15 x 8 cm, the weight is 600 g and it is powered by a 9 V battery. We have characterised the responsiveness of the sensor using calibrated sucrose solutions and were able to measure changes as small as 3.3 x 10(-6) refractive index units. To demonstrate functionality of the sensor, we have prepared surfaces with an antibody fragment specific for the biological toxin ricin. We were able to detect ricin at 200 ng/mL in 10 min, which is approximately 2500 times less than the minimum lethal dose. We were also able to verify positive binding within a second 10 min window. This sensor demonstrates important steps required for the development of fully integrated, hand-held sensor devices and will form the basis of a multi-analyte system, to be developed in the near future. It also represents the first completely hand-held SPR device, not requiring external power or a computer connection to operate.


Subject(s)
Biosensing Techniques/instrumentation , Environmental Monitoring/instrumentation , Hazardous Substances/analysis , Ricin/analysis , Surface Plasmon Resonance/instrumentation , Biosensing Techniques/methods , Environmental Monitoring/methods , Equipment Design , Equipment Failure Analysis , Miniaturization , Reproducibility of Results , Sensitivity and Specificity
4.
Clin Exp Allergy ; 37(8): 1189-98, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17651149

ABSTRACT

BACKGROUND: Back-titration of inhaled corticosteroid (ICS) dose in well-controlled asthma patients is emphasized in clinical guidelines, but there are few published data on the airway cell and cytokine changes in relation to ICS reduction. In our study, 20 mild-to-moderate persistent (inspite of low-moderate dose ICS treatment) asthmatic subjects prospectively rendered largely asymptomatic by high-dose ICS were assessed again by clinical, physiological, and airway inflammatory indices after 4-8 weeks of reduced ICS treatment. We aimed at assessing the underlying pathological changes in relation to clinical deterioration. METHODS: Patients recorded daily symptom scores and peak expiratory flows (PEF). Spirometry and airways hyperreactivity (AHR) were measured and bronchoscopy was performed with assessment of airway biopsies (mast cells, eosinophils, neutrophils, and T lymphocytes), bronchoalveolar lavage (BAL) IL-5 and eotaxin levels and cellular profiles at the end of high-dose ICS therapy and again after ICS dose reduction. Baseline data were compared with symptomatic steroid-free asthmatics (n=42) and non-asthmatic controls (n=28). RESULTS: After ICS reduction, subjects experienced a variable but overall significant increase in symptoms and reductions in PEF and forced expiratory volume in 1 s. There were no corresponding changes in AHR or airways eosinophilia. The most relevant pathogenic changes were increased CD4(+)/CD8(+) T cell ratio, and decreased sICAM-1 and CD18 macrophage staining (potentially indicating ligand binding). However, there was no relationship between the spectrum of clinical deterioration and the changes in cellular profiles or BAL cytokines. CONCLUSIONS: These data suggest that clinical markers remain the most sensitive measures of early deterioration in asthma during back-titration of ICS, occurring at a time when AHR and conventional indices of asthmatic airway inflammation appear unchanged. These findings have major relevance to management and to how back-titration of ICS therapy is monitored.


Subject(s)
Asthma/metabolism , Chemokines, CC/metabolism , Interleukin-5/metabolism , Leukocytes/metabolism , Pulmonary Eosinophilia/metabolism , Adrenal Cortex Hormones/administration & dosage , Adult , Aged , Asthma/drug therapy , Asthma/pathology , Asthma/physiopathology , Biomarkers/metabolism , Biopsy , Bronchoalveolar Lavage Fluid , Chemokine CCL11 , Female , Humans , Leukocytes/pathology , Male , Middle Aged , Monitoring, Physiologic , Peak Expiratory Flow Rate/drug effects , Pulmonary Eosinophilia/drug therapy , Pulmonary Eosinophilia/pathology , Pulmonary Eosinophilia/physiopathology , Spirometry
5.
Thorax ; 62(4): 314-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17105777

ABSTRACT

BACKGROUND: Subepithelial hypervascularity and angiogenesis in the airways are part of structural remodelling of the airway wall in asthma, but the effects of inhaled corticosteroids (ICS) on these have not been explored. Increased vascularity in asthma may contribute to a number of functional abnormalities. A study was undertaken to explore angiogenic modulation by ICS and its likely regulation via vascular endothelial growth factor (VEGF), its receptors and the angiopoietins. METHODS: A placebo-controlled intervention study with ICS in asthma was performed, examining vascularity, VEGF, its receptors (VEGFR1 and VEGFR2), and angiopoietin-1 (Ang1) to assess which of these factors were changed in the asthmatic airways after ICS treatment. Airway wall biopsy specimens, lavage fluid and cells were obtained from 35 patients with mild asthma randomised to receive ICS or placebo for 3 months, after which bronchoscopic examination and sample collection were repeated. Immunohistochemistry and image analysis were used to obtain quantitative measures of vessels, angiogenic sprouts, VEGF, VEGFR1, VEGFR2 and Ang1 staining in airway biopsy specimens. ELISA was used to assess VEGF concentrations in the lavage fluid. RESULTS: Vessel, VEGF and sprout staining were decreased after 3 months of ICS treatment. VEGF levels remained unchanged. VEGF receptors and Ang1 staining were not reduced after treatment. CONCLUSIONS: The findings of this study support an effect of ICS in downregulating angiogenic remodelling in the airways in asthma, associated with decreasing VEGF activity within the airway wall. The environment of the airways after treatment with ICS, with changes in the balance between VEGF, its receptors, Ang1 and sprouts, appears to be less angiogenic than in untreated asthma.


Subject(s)
Androstadienes/administration & dosage , Anti-Asthmatic Agents/administration & dosage , Asthma/drug therapy , Bronchi/blood supply , Vascular Endothelial Growth Factor A/metabolism , Administration, Inhalation , Adult , Aged , Androstadienes/adverse effects , Anti-Asthmatic Agents/adverse effects , Fluticasone , Humans , Metered Dose Inhalers , Middle Aged , Neovascularization, Pathologic/chemically induced
6.
Clin Exp Allergy ; 35(12): 1565-71, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16393322

ABSTRACT

BACKGROUND: Asthma is accepted as a disease characterized by airway inflammation, with evidence that airway structural changes, or 'remodelling' occurs. There are few studies relating airway physiology, inflammation and remodelling, however. We have carried out a study of inter-relationships between airway inflammation, airway remodelling, reticular basement membrane (RBM) thickening, and bronchial hyper-reactivity (BHR), before and after high-dose inhaled corticosteroid (fluticasone propionate 750 microg b.d.), in a group of relatively mild but symptomatic, steroid naïve asthma patients. METHODS: Double-blind, randomized, placebo-controlled, parallel group study of inhaled corticosteroid (ICS) in 35 asthmatics, with bronchoalveolar lavage (BAL) and airway endobronchial biopsy (EBB) for inflammatory cell profiles and EBB for airway remodelling carried out at baseline, 3 and 12 months. RESULTS: At baseline RBM thickening was related to BAL mast cells and EBB eosinophil counts. In turn baseline log EBB EG2 eosinophil count, log%BAL epithelial cells and log RBM thickness explained 55% of the variability in BHR. CONCLUSION: We provide new information that airway inflammation, remodelling, and BHR in asthma are inter-related and improved by ICS therapy. Our data potentially support the need for early and long-term intervention with ICS even in relatively mild asthmatics, and the need to further assess the potential merit of longitudinal BHR testing in management of some patients, as this may reflect both airway inflammation and remodelling.


Subject(s)
Asthma/immunology , Asthma/pathology , Bronchi/immunology , Bronchi/pathology , Hypersensitivity/immunology , Hypersensitivity/pathology , Basement Membrane/immunology , Basement Membrane/pathology , Biopsy , Bronchial Hyperreactivity/chemically induced , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/pathology , Bronchial Provocation Tests , Bronchoalveolar Lavage Fluid/chemistry , Bronchoconstrictor Agents , Case-Control Studies , Humans , Methacholine Chloride , Regression Analysis , Reticulin/immunology , Spirometry
7.
J Surg Res ; 107(1): 101-7, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12384070

ABSTRACT

An attenuated (DeltacyA, Deltacrp) strain of Salmonella typhimurium (chi4550) containing a gene for human IL-2 (chi4550pIL2) reduces hepatic tumor burden when orally inoculated into mice with liver cancer; however, wild-type S. typhimurium is also associated with cancer regression. Therefore, experiments were designed to clarify the invasiveness and the anti-tumor properties of three strains of S. typhimurium. S. typhimurium chi4550pIL2, chi4550, or wild type (WT) was incubated with mature Caco-2 and HT-29 enterocytes, and S. typhimurium internalization was assessed. For infectivity experiments, mice were orally inoculated with saline or 10(9)S. typhimurium chi4550pIL2, chi4550, or WT; 48 h later mice were sacrificed for analysis of cecal bacteria and S. typhimurium translocation to mesenteric lymph nodes. For experiments involving tumor implantation, four groups were studied: saline control, tumor alone, chi4550pIL2+tumor, and chi4550+tumor. Mice were orally inoculated with saline or S. typhimurium and underwent laparotomy 24 h later with 5 x 10(4) MCA38 murine adenocarcinoma cells injected into the spleen. On day 14, liver tumors were counted and peripheral blood and hepatic lymphocyte populations were analyzed by FACScan. Attenuated S. typhimurium exhibited decreased internalization by cultured enterocytes and decreased infectivity after oral inoculation. Mice treated with chi4550pIL2 or chi4550 had fewer liver tumors and increased populations of hepatic and circulating NK1.1(+)CD3(-) lymphocytes compared to mice treated with saline (P < 0.01). These data suggest that attenuated S. typhimurium may have an application as an anti-tumor agent.


Subject(s)
Blood Cells/pathology , Liver Neoplasms/microbiology , Liver Neoplasms/pathology , Liver/pathology , Salmonella Infections, Animal/pathology , Salmonella typhimurium , Animals , Antigens/analysis , Antigens, Ly , Antigens, Surface , Blood Cells/immunology , CD3 Complex/analysis , Caco-2 Cells , Cells, Cultured , Enterocytes/microbiology , Female , HT29 Cells , Humans , Lectins, C-Type , Liver/immunology , Lymphocytes/physiology , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily B , Phenotype , Proteins/analysis , Salmonella Infections, Animal/immunology , Salmonella typhimurium/physiology
8.
Thorax ; 57(4): 309-16, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11923548

ABSTRACT

BACKGROUND: There are few data in asthma relating airway physiology, inflammation and remodelling and the relative effects of inhaled corticosteroid (ICS) treatment on these parameters. A study of the relationships between spirometric indices, airway inflammation, airway remodelling, and bronchial hyperreactivity (BHR) before and after treatment with high dose inhaled fluticasone propionate (FP 750 microg bd) was performed in a group of patients with relatively mild but symptomatic asthma. METHODS: A double blind, randomised, placebo controlled, parallel group study of inhaled FP was performed in 35 asthmatic patients. Bronchoalveolar lavage (BAL) and airway biopsy studies were carried out at baseline and after 3 and 12 months of treatment. Twenty two normal healthy non-asthmatic subjects acted as controls. RESULTS: BAL fluid eosinophils, mast cells, and epithelial cells were significantly higher in asthmatic patients than in controls at baseline (p<0.01). Subepithelial reticular basement membrane (rbm) thickness was variable, but overall was increased in asthmatic patients compared with controls (p<0.01). Multiple regression analysis explained 40% of the variability in BHR, 21% related to rbm thickness, 11% to BAL epithelial cells, and 8% to BAL eosinophils. The longitudinal data corroborated the cross sectional model. Forced expiratory volume in 1 second improved after 3 months of treatment with FP with no further improvement at 12 months. PD(20) improved throughout the study. BAL inflammatory cells decreased following 3 months of treatment with no further improvement at 12 months (p<0.05 v placebo). Rbm thickness decreased in the FP group, but only after 12 months of treatment (mean change -1.9, 95% CI -3 to -0.7 microm; p<0.01 v. baseline, p<0.05 v. placebo). A third of the improvement in BHR with FP was associated with early changes in inflammation, but the more progressive and larger improvement was associated with the later improvement in airway remodelling. CONCLUSION: Physiology, airway inflammation and remodelling in asthma are interrelated and improve with ICS. Changes are not temporally concordant, with prolonged treatment necessary for maximal benefit in remodelling and PD(20). Determining the appropriate dose of inhaled steroids only by reference to symptoms and lung function, as specified in current international guidelines, and even against indices of inflammation may be over simplistic. The results of this study support the need for early and long term intervention with ICS, even in patients with relatively mild asthma.


Subject(s)
Androstadienes/therapeutic use , Anti-Inflammatory Agents/therapeutic use , Asthma/pathology , Basement Membrane/pathology , Bronchodilator Agents/therapeutic use , Administration, Topical , Adult , Aged , Asthma/drug therapy , Asthma/physiopathology , Biopsy/methods , Bronchial Hyperreactivity/drug therapy , Bronchial Hyperreactivity/pathology , Bronchial Hyperreactivity/physiopathology , Bronchitis/pathology , Bronchitis/physiopathology , Bronchoalveolar Lavage Fluid/cytology , Bronchoscopy/methods , Collagen/analysis , Cross-Sectional Studies , Double-Blind Method , Fluticasone , Forced Expiratory Volume/drug effects , Glucocorticoids , Humans , Longitudinal Studies , Mast Cells , Middle Aged , Observer Variation , Regression Analysis
9.
Am J Respir Crit Care Med ; 164(9): 1718-21, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11719315

ABSTRACT

The airways of individuals with asthma are less distensible than normal and it has been assumed that this may be due to airway remodeling associated with chronic inflammation, although there are currently no available data directly relating these two aspects of asthma. We have therefore carried out a study of the relationship between airway distensibility (DeltaVD) and subepithelial reticular basement membrane (RBM) thickening as an index of airway remodeling, in a group of patients with relatively mild but symptomatic asthma. Our methods included a cross-sectional study of DeltaVD in patients with mild to moderate atopic asthma, with matched airway biopsy for structural components. We confirmed that DeltaVD was lower in patients with asthma than in normal individuals (19.8 +/- 1.1 versus 24.1 +/- 1.5; p < 0.05) and that RBM thickness was increased in patients with asthma (9.1 +/- 2.2 versus 7.7 +/- 1.2 microm; p < 0.01). There was a negative correlation between DeltaVD and RBM thickness in asthma (r = -0.37, p = 0.03) and positive correlations between percent predicted postbronchodilator large and small airway function (for percent predicted FEV(1 )versus DeltaVD, r = 0.59, p < 0.001). We conclude that, cross-sectionally, DeltaVD was related to airway remodeling (RBM thickening) and airflow limitation (percent predicted large and small airway function). Our findings support the hypothesis that DeltaVD is a physiologic test that is reflective of airway remodeling.


Subject(s)
Airway Obstruction/pathology , Asthma/pathology , Adult , Aged , Airway Obstruction/etiology , Airway Obstruction/physiopathology , Asthma/complications , Asthma/physiopathology , Basement Membrane/pathology , Case-Control Studies , Cross-Sectional Studies , Female , Forced Expiratory Flow Rates , Forced Expiratory Volume , Humans , Least-Squares Analysis , Linear Models , Male , Middle Aged
10.
J Surg Res ; 97(1): 97-102, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11319888

ABSTRACT

BACKGROUND: Clostridium difficile toxins alter permeability in cultured enterocytes and may alter intestinal epithelial permeability to bacteria in vivo. Experiments were designed to test the effects of C. difficile toxins on in vitro interactions of Enterococcus gallinarum with cultured enterocytes, as well as on translocation of E. gallinarum in mice. MATERIALS AND METHODS: Mature Caco-2 and HT-29 enterocytes were pretreated with C. difficile toxin A or toxin B followed by incubation with E. gallinarum. E. gallinarum-enterocyte interactions were assessed by quantitative culture. For in vivo experiments, antibiotic-treated mice were orally inoculated with C. difficile or saline, and all mice were orally inoculated 24 h later with E. gallinarum and sacrificed after another 24 h for analysis of cecal bacteria, cecal C. difficile toxin, and enterococcal translocation. Cecal C. difficile toxin was assayed as cytopathic effects on human foreskin fibroblasts. RESULTS: Although neither toxin had a noticeable effect on bacterial internalization by cultured enterocytes, C. difficile toxins were associated with increased E. gallinarum transmigration across confluent enterocyte cultures. Mice orally inoculated with saline rather than C. difficile (n = 29) had no detectable cecal toxin, while mice orally inoculated with C. difficile (n = 30) had detectable cecal toxin. Viable E. gallinarum was recovered from the mesenteric lymph nodes of 97% of mice orally inoculated with saline followed by oral E. gallinarum, but only 37% of mice orally inoculated with C. difficile followed by oral E. gallinarum (P < 0.01). CONCLUSIONS: These results suggested that observations with cultured enterocytes, demonstrating that C. difficile toxins facilitated bacterial migration across the intestinal epithelium, might have little in vivo relevance in a mouse model of antibiotic-induced C. difficile overgrowth.


Subject(s)
Bacterial Proteins , Bacterial Toxins/pharmacology , Bacterial Translocation , Enterococcus/physiology , Enterotoxins/pharmacology , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Bacterial Toxins/analysis , Cecum/microbiology , Cell Line , Cell Membrane Permeability , Clostridioides difficile/pathogenicity , Enterocytes/drug effects , Enterocytes/metabolism , Enterocytes/microbiology , Enterotoxins/analysis , Female , Gentamicins/pharmacokinetics , Gentamicins/pharmacology , Humans , Intestinal Mucosa/metabolism , Intestines/anatomy & histology , Lymph Nodes/microbiology , Mesentery , Mice
11.
Shock ; 14(6): 629-34, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11131913

ABSTRACT

Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis. We hypothesized that C. difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier. Experiments were designed to clarify the effects of C. difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions. In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL). To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles. Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers. To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers. Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes. Thus intestinal colonization with toxigenic C. difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.


Subject(s)
Bacterial Proteins , Bacterial Toxins/toxicity , Enterocytes/drug effects , Enterocytes/microbiology , Enterotoxins/toxicity , Actins/metabolism , Bacterial Adhesion , Cell Survival , Clostridioides difficile/pathogenicity , Enterocytes/physiology , HT29 Cells , Humans , Microscopy, Electron, Scanning , Permeability
12.
Arch Surg ; 134(11): 1235-41; discussion 1241-2, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10555639

ABSTRACT

BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium. HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria. DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration. CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.


Subject(s)
Bacterial Proteins , Bacterial Toxins , Bacterial Translocation/physiology , Clostridioides difficile/physiology , Enterotoxins/physiology , Intestinal Mucosa/microbiology , Bacterial Adhesion , Enterocytes/physiology , Enterocytes/ultrastructure , Humans
13.
Clin Transplant ; 12(3): 278-80, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9642524

ABSTRACT

Gallbladder perforation is a frequent complication of acute acalculous cholecystitis (AAC), resulting in substantially increased morbidity and mortality. Two groups of patients are at increased risk for perforation: those with systemic diseases (especially peripheral vascular disease, intrinsic heart disease, or diabetes) and those who are chronically immunosuppressed. The current population of solid organ transplant recipients meets both criteria. We describe an unusual case of gallbladder perforation as a complication of AAC in an otherwise healthy kidney transplant recipient. Because transplant recipients are at increased risk for gallbladder perforation, maintaining a high index of suspicion for this complication will help avoid the increased morbidity and mortality associated with this diagnosis.


Subject(s)
Cholecystitis/complications , Cholecystitis/diagnostic imaging , Gallbladder Diseases/etiology , Acute Disease , Cholangiopancreatography, Endoscopic Retrograde , Cholecystectomy , Cholecystitis/pathology , Cholecystitis/surgery , Gallbladder Diseases/diagnostic imaging , Glomerulonephritis/surgery , Humans , Kidney Transplantation , Male , Middle Aged , Tomography, X-Ray Computed
14.
Gastroenterology ; 110(5): 1429-37, 1996 May.
Article in English | MEDLINE | ID: mdl-8613048

ABSTRACT

BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis has been associated with diarrheal disease, and the enterotoxin has a cytopathic effect on cultured HT-29 enterocytes. Experiments were designed to determine the effect of B. fragilis enterotoxin on bacteria-enterocyte interactions. METHODS: Confluent HT-29 enterocytes were incubated for 1 hour with B. fragilis enterotoxin, followed by 1 hour of incubation with pure cultures of enteric bacteria, namely, Salmonella typhimurium (two strains), Listeria monocytogenes (three strains), Proteus mirabilis, Escherichia coli (three strains), and Enterococcus faecalis. Enterocyte viability was assessed using vital dyes, epithelial permeability was measured using transepithelial electrical resistance, enterocyte morphology and bacteria-enterocyte interactions were visualized using light and electron microscopy, and bacterial internalization was assessed using a quantitative culture of lysed enterocytes. RESULTS: B. fragilis enterotoxin did not affect enterocyte viability but decreased transepithelial electrical resistance, and individual enterocytes pulled apart. Enterotoxin pretreatment decreased internalization of L. monocytogenes (P < 0.01) but increased (P < 0.01) internalization of the other strains of enteric bacteria. Augmented bacterial internalization was associated with preferential bacterial adherence on the exposed lateral surface of enterotoxin-treated enterocytes. CONCLUSIONS: B. fragilis enterotoxin was associated with HT-29 cell rounding and with augmented internalization of selected strains of enteric bacteria that were preferentially adherent on the exposed enterocyte lateral surface.


Subject(s)
Bacterial Physiological Phenomena , Bacteroides fragilis/metabolism , Enterotoxins/pharmacology , Intestines/cytology , Intestines/microbiology , Metalloendopeptidases/pharmacology , Bacterial Adhesion , Cell Membrane Permeability , Cell Survival , Cells, Cultured , Electric Impedance , Epithelium/metabolism , Humans , Intestinal Mucosa/metabolism , Microscopy, Electron , Microscopy, Electron, Scanning
15.
Shock ; 2(1): 29-33, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7735981

ABSTRACT

Genetically macrophage-deficient op/op mice have a total absence of macrophage colony-stimulating factor (also known as colony-stimulating factor 1 or CSF-1), and therefore an absence of a population of macrophages dependent on CSF-1. op/op mice also have profound secondary deficiencies in certain cytokines secreted by this macrophage population, such as tumor necrosis factor, interleukin-1, and granulocyte colony-stimulating factor. In the present study, op/op mice were used to clarify the role of the macrophage in two clinical processes: (a) bacterial translocation in response to antibiotic-induced intestinal overgrowth, and (b) endotoxin-induced bacterial translocation, morbidity, and mortality. The results were unexpected, in that bacterial translocation and endotoxin-induced morbidity and mortality were similar in op/op mice and their functionally normal littermates. These data indicated either that a specific macrophage population and its cytokines (including tumor necrosis factor and interleukin 1) might not play pivotal roles in the pathogenesis of bacterial translocation and endotoxin-induced septic shock, or alternatively, as yet unknown redundancies in vivo might compensate for the genetic deficiencies associated with the op/op mutation.


Subject(s)
Escherichia coli Infections/mortality , Lipopolysaccharides , Macrophages/metabolism , Shock, Septic/mortality , Animals , Anti-Bacterial Agents/pharmacology , Escherichia coli/growth & development , Escherichia coli/isolation & purification , Escherichia coli Infections/metabolism , Escherichia coli Infections/physiopathology , Macrophage Colony-Stimulating Factor/deficiency , Macrophages/pathology , Mice , Mice, Mutant Strains , Shock, Septic/metabolism , Shock, Septic/physiopathology
16.
J Med Microbiol ; 38(5): 345-53, 1993 May.
Article in English | MEDLINE | ID: mdl-8487291

ABSTRACT

The purpose of this study was to clarify the association between the oral infectivity of a bacterial strain and its susceptibility to ingestion by mononuclear phagocytes or ability to survive within them. Ten bacterial strains tested--all of known oral infectivity--comprised Salmonella typhimurium, Listeria monocytogenes (three strains), Escherichia coli (two strains), Proteus mirabilis, Enterococcus faecalis, Bacteroides fragilis, and a Bacteroides sp. The phagocytic uptake of each strain was measured as the bacteria to phagocyte ratio after mononuclear phagocytes in mouse peritoneal exudate were permitted to ingest bacteria in vivo for 3 min. The three Listeria strains were the most susceptible to phagocytic uptake and the Salmonella strain was relatively resistant. The intracellular survival of each strain was studied during a subsequent 2 h in-vitro incubation of the mononuclear phagocytes that had been permitted to ingest bacteria in vivo. The strains with the best intracellular survival were Ent. faecalis and two of the three Listeria strains. The ability of S. typhimurium to survive intracellularly was intermediate but better than that of the two E. coli strains. Oral infectivity was not consistently correlated with susceptibility to ingestion by mononuclear phagocytes or ability to survive within them.


Subject(s)
Bacteria/pathogenicity , Mouth/microbiology , Phagocytes/metabolism , Phagocytosis , Animals , Female , Hemolysin Proteins/metabolism , Listeria monocytogenes/growth & development , Listeria monocytogenes/pathogenicity , Mice , Salmonella typhimurium/growth & development , Salmonella typhimurium/pathogenicity
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