Increased levels of enzymes involved in local estradiol synthesis in chronic obstructive pulmonary disease.

INTRODUCTION: Steroid hormones are involved in lung development, pulmonary inflammation, and lung cancer. Estrogen signaling and exposure may play a role in pulmonary disorders, including COPD. In both genders, estrogens can be generated locally in the lungs and this contributes importantly to the tissue exposure to these steroids. OBJECTIVE: To characterize and assess differences in localization of estrogen receptors and enzymes involved in the local generation of estrogens in COPD. METHODS: Estrogen Receptor alpha (ERα/ESR1), Estrogen Receptor beta (ERß/ESR2) and G-protein-coupled estrogen receptor 1 (GPER) were explored by real-time (RT)-PCR analysis (mRNA expression), immunohistochemistry and western blotting in controls and COPD patients. mRNA expression of the enzymes involved in the local estrogen generation - i.e. aromatase (CYP19A1), 17beta-hydroxysteroid dehydrogenases (17ß-HSDs) 1, 2, 4, 5, 7 and 12, steroid sulfatase (STS) and sulfotransferase (SULT1E1) - were analyzed by RT-PCR. RESULTS: ERα, ERß and GPER were expressed in lung tissue, but no differences were observed between patients and controls. The main enzymes involved in local estrogen generation were also present in both normal and COPD lung tissue. In lungs of COPD patients compared with controls, we observed increased expression of the enzymes 17ß-HSD type 1 and aromatase (positive association), both involved in the local synthesis of active estrogens. CONCLUSION: All ER subtypes are present in the lung. The shift in local mRNA level of estrogen metabolic enzymes suggests that exposure to estrogens is involved in the pathogenesis of COPD.

Enhanced pulmonary leptin expression in patients with severe COPD and asymptomatic smokers.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by an abnormal inflammatory reaction of the lungs involving activation of epithelial cells. Leptin is a pleiotropic cytokine important in the regulation of immune responses via its functional receptor Ob-Rb. This study was undertaken to test the hypothesis that severe COPD is associated with increased leptin expression in epithelial cells. METHODS: Immunohistochemistry for leptin was performed on peripheral lung specimens from 20 patients with COPD (GOLD stage 4), 14 asymptomatic ex-smokers and 13 never smokers. Leptin and Ob-Rb mRNA expression were determined by rtPCR in cultured primary bronchial epithelial cells and primary type II pneumocytes. NCI-H292 and A549 cell lines were used to study functional activation of leptin signalling. RESULTS: Leptin immunoreactivity in lung tissue was observed in bronchial epithelial cells, type II pneumocytes, macrophages (tissue/alveolar) and interstitial lymphocytic infiltrates. rtPCR analysis confirmed pulmonary leptin and Ob-Rb mRNA expression in primary bronchial epithelial cells and pneumocytes. Leptin-expressing bronchial epithelial cells and alveolar macrophages were markedly higher in patients with severe COPD and ex-smokers than in never smokers (p<0.02). Exposure of cultured primary bronchial epithelial cells to smoke resulted in increased expression of both leptin and Ob-Rb (p<0.05). Leptin induced phosphorylation of STAT3 in both NCI-H292 and A549 cells. CONCLUSIONS: Leptin expression is increased in bronchial epithelial cells and alveolar macrophages of ex-smokers with or without severe COPD compared with never smokers. A functional leptin signalling pathway is present in lung epithelial cells.

Myeloperoxidase modulates lung epithelial responses to pro-inflammatory agents.

During extensive inflammation, neutrophils undergo secondary necrosis causing myeloperoxidase (MPO) release that may damage resident lung cells. Recent observations suggest that MPO has pro-inflammatory properties, independent of its enzymatic activity. The aims of the present study were to characterise MPO internalisation by lung epithelial cells and to investigate the effect of MPO on oxidative stress, DNA damage and cytokine production by lung epithelial cells. Human alveolar and bronchial epithelial cells were stimulated with MPO, with or without priming the cells with pro-inflammatory stimuli. MPO protein was detected in the cell cytoplasm. Expression of haemoxygenase (HO)-1 and DNA strand breakage were determined. The production of interleukin (IL)-8 and -6 were measured. Analyses of MPO-stimulated cells demonstrated MPO presence in the cells. HO-1 expression was increased after MPO stimulation and increased further when cells were primed before MPO stimulation. MPO exposure also induced DNA strand breakage. Interestingly, MPO inhibited IL-8 production in bronchial, but not alveolar epithelium. In conclusion, alveolar and bronchial epithelial cells can internalise myeloperoxidase. Stimulation with myeloperoxidase increases haemoxygenase-1 expression and DNA strand breakage, suggesting cell damaging capacity of myeloperoxidase. In addition, myeloperoxidase inhibited interleukin-8 production by bronchial epithelial cells, indicating a negative feedback loop for neutrophil recruitment.

Breath condenser coatings affect measurement of biomarkers in exhaled breath condensate.

Exhaled breath condensate collection is not yet standardised and biomarker measurements are often close to lower detection limits. In the current study, it was hypothesised that adhesive properties of different condenser coatings interfere with measurements of eicosanoids and proteins in breath condensate. In vitro, condensate was derived from a collection model using two test solutions (8-isoprostane and albumin) and five condenser coatings (silicone, glass, aluminium, polypropylene and Teflon). In vivo, condensate was collected using these five coatings and the EcoScreen condenser to measure 8-isoprostane, and three coatings (silicone, glass, EcoScreen) to measure albumin. In vitro, silicone and glass coatings had significantly higher albumin recovery compared with the other coatings. A similar trend was observed for 8-isoprostane recovery. In vivo, median (interquartile range) 8-isoprostane concentrations were significantly higher using silicone (9.2 (18.8) pg.mL(-1)) or glass (3.0 (4.5) pg.mL(-1)) coating, compared with aluminium (0.5 (2.4) pg.mL(-1)), polypropylene (0.5 (0.5) pg.mL(-1)), Teflon (0.5 (0.0) pg.mL(-1)), and EcoScreen (0.5 (2.0) pg.mL(-1)). Albumin in vivo was mainly detectable using glass coating. In conclusion, a condenser with silicone or glass coating is more efficient for measurement of 8-isoprostane or albumin in exhaled breath condensate, than EcoScreen, aluminium, polypropylene or Teflon. Guidelines for exhaled breath condensate standardisation should include the most valid condenser coating to measure a specific biomarker.

Enhanced levels of hyaluronan in lungs of patients with COPD: relationship with lung function and local inflammation.

BACKGROUND: Chronic inflammation and airway remodelling are characteristics of chronic obstructive pulmonary disease (COPD). Hyaluronan (HA) is an extracellular matrix compound with proinflammatory activity. HA levels in induced sputum from patients with COPD were measured and related to local inflammation. The expression of hyaluronan synthase 2 (HAS2) and hyaluronidase 2 (HYAL2) was analysed in lung tissue. METHODS: Sputum was obtained from 18 patients with COPD (forced expiratory volume in 1 second (FEV(1)) 62% predicted (range 20-76)) and 14 healthy smokers. HA and inflammatory markers were measured using ELISA assays. Lung sections were obtained from five patients with severe COPD (FEV(1) <30%) and from five smokers, and mRNA levels of HAS2 and HYAL2 were analysed by polymerase chain reaction. RESULTS: HA levels were significantly higher in the sputum from patients with COPD than controls. The COPD population appeared to consist of two subpopulations with either high or moderate HA levels. The subgroup of patients with high HA levels had lower FEV(1) than the moderate HA group. In addition, neutrophil influx and levels of interleukin-8, and the soluble tumour necrosis factor receptors R55 and R75 were significantly higher in patients with high HA levels than in those with moderate HA levels and controls. Semiquantitative analysis revealed enhanced expression of HYAL2 in lung tissue of patients with severe COPD compared with control subjects. CONCLUSION: These data indicate a relationship between HA levels, local inflammation and severity of disease, and suggest enhanced breakdown of HA in the lungs of patients with COPD.

Leptin as local inflammatory marker in COPD.

INTRODUCTION: Chronic inflammation of the lung is a characteristic finding in chronic obstructive pulmonary disease (COPD). Leptin is a pleiotropic cytokine thought to play a role in host response to inflammation. As recent studies have shown that leptin receptors are present in the lung, this study aimed to determine if leptin is detectable in induced sputum of COPD patients and if there is a relationship between leptin and other inflammatory markers in sputum. METHODS: Sputum was induced in 14 male patients with moderate COPD (FEV1: 56 (15) % pred.). Leptin, total tumour necrosis factor (TNF)-alpha, and C-reactive protein (CRP) were analyzed in induced sputum supernatant by ELISA. Leptin was also determined in EDTA plasma. RESULTS: Leptin was detectable in induced sputum of 10 COPD patients. A significant relationship was found between sputum leptin and CRP (r = 0.943, P < 0.001) and total TNF-alpha (r = 0.690, P < 0.01). Plasma leptin and sputum leptin were inversely correlated (r = -0.643, P < 0.01). CONCLUSION: The present study demonstrated that leptin is detectable in induced sputum of patients with moderate COPD and is related to other inflammatory markers. The observed correlations between leptin and inflammatory markers in sputum may indicate that leptin is involved in the local inflammatory response in COPD.

Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells: no role for tumor necrosis factor-alpha and infiltrating neutrophils.

This study investigated apoptosis in lungs after local exposure to lipopolysaccharide (LPS). Mice were instilled intratracheally with 5 microg LPS, which corresponds to the amount acquired by smoking approximately 25 cigarettes, and killed at different time points after exposure. Our data demonstrate that local LPS exposure resulted in apoptosis in lungs from 2 h and peaked at 24 h, as detected by ligation-mediated polymerase chain reaction. Morphologic examination and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end label staining demonstrated apoptosis in bronchial epithelial cells early after intratracheal (IT) LPS challenge, whereas infiltrating neutrophils displayed positive staining at 24 and 72 h after exposure. Apoptosis in lungs clearly preceded pulmonary neutrophil infiltration, confirming that neutrophils did not contribute to pulmonary apoptosis at early time points. Further, using three experimental approaches--namely, anti-tumor necrosis factor (TNF)-alpha treatment, IT TNF-alpha instillation, and TNF/lymphotoxin-alpha knockout mice--we demonstrate that TNF-alpha, which was elevated in lungs at both messenger RNA and protein levels after IT LPS challenge, was no primary mediator in LPS-induced apoptosis at early time points. Thus, local LPS exposure in mice resulted in early apoptosis of bronchial epithelial cells independent of infiltrating neutrophils and TNF-alpha, which suggests that apoptosis of bronchial epithelium may be involved in airway injury during exposure to LPS.

Production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to bacterial endotoxins.

This study demonstrates for the first time that respiratory epithelial cells are able to produce the acute phase protein lipopolysaccharide (LPS)-binding protein (LBP), which is known to play a central role in the defense to bacterial endotoxins (or LPS). Indications for local presence of LBP in human lung was obtained via reverse transcriptase/polymerase chain reaction that showed LBP messenger RNA (mRNA) expression. Therefore, LBP production by the human lung epithelial cell line A549, a human adenocarcinoma with features of type II pneumocytes, was studied. These cells produced LBP in response to interleukin (IL)-1beta, IL-6, and tumor necrosis factor- alpha, a response that was strongly enhanced by dexamethasone. In addition, LBP mRNA was detected in A549 cells, in increasing amounts as a result of stimulation. The pattern of cytokine-induced LBP production in A549 cells was similar to the pattern in the human liver epithelial cell line HuH-7. Moreover, the molecular weight of A549-derived LBP was approximately 60 kD, which is similar to HuH-7-derived LBP. Biologic activity of LBP produced by A549 cells was evaluated on the basis of its ability to interact with LPS. Further indications that type II alveolar epithelial cells are able to produce LBP were obtained from the observations that the murine lung type II epithelial cell line C10 produced murine LBP, and that isolated human primary type II pneumocytes expressed LBP mRNA, which was enhanced after stimulation of cells. The local production of this endotoxin binding protein by lung epithelial cells might contribute to a highly specific response at the site of exposure to bacteria and bacterial endotoxins.

Introduction, persistence and fade-out of porcine reproductive and respiratory syndrome virus in a Dutch breeding herd: a mathematical analysis.

The objective of this study was to investigate the dynamics of PRRSV infection and to quantify transmission within a breeding herd, and its impact on herd performance. For this purpose a longitudinal study was performed in a closed breeding herd of 115 sows. Statistical methods and Monte Carlo simulations based on stochastic SIR models were used to analyse the observational data. Moreover, a case-control study was performed to determine whether seroconversion of sows during gestation was associated with aberrant litters. The transmission parameter R was estimated to be 3.0 (95% confidence interval 1.5-6.0) for the model version based on the most plausible assumptions that the infectious period lasts 56 days and no lifelong immunity exists after infection. Based on simulations using a breeding herd of equal size the average time-to-extinction was estimated to be 6 years; using a herd of twice the size, it was 80 years. Furthermore, in contrast to the epidemic phase of the disease, the endemic phase was not detrimental to herd performance.

Risk of recurrence of repeat breeding in sows with a repeat breeding in the first parity.

Risk of recurrence of repeat breeding in sows with a repeat breeding in the 1st parity was investigated in 241 sow herds (1985-1990). The association between repeat breeding in the 1st and subsequent parities was examined using data on 9,120 sows. Each sow contributed only one pair of observations to the total data set. Occurrence of repeat breeding in the 1st and higher parities was presented in terms of recurrent risk rates (rRR), adjusted for different strata of herds. Sows with a repeat breeding in the 1st parity did not have a significantly higher risk rate for recurrence of a repeat breeding in the 2nd parity (rRR = 1.12, 95% confidence interval .97-1.29) and in the 3rd parity (rRR = 1.10, 95% confidence interval .86-1.41) compared to sows without a repeat breeding in the 1st parity. Sows with a repeat breeding in the 1st parity had a slightly higher risk rate for recurrence of a repeat breeding in the 4th or higher parity (rRR = 1.07, 95% confidence interval 1.03-1.10), compared to sows without a repeat breeding in the 1st parity. However, this risk is, in a biological sense, very small.

The clinical recovery of fattening pigs from respiratory disease after treatment with two injectable oxytetracycline formulations.

A double blind randomized clinical trial was performed with pigs suffering from clinical respiratory disease. The goal of the trial was to test the null hypothesis that the clinical recovery after treatment with two oxytetracycline injectables with different pharmacokinetic profiles (high peak concentration and low persistence versus low peak concentrations and long persistence) was similar. Fattening pigs (n = 529) were treated intramuscularly with either product A or product B at a dose of 20 mg OTC per kg b.w. when they showed signs of acute pneumonia, i.e., coughing, tachypnoea or dyspnoea combined with a rectal temperature of 40 degrees C or higher. When necessary, treatment was given again after 3 and/or 6 days. Both treatments resulted in a rapid fall in mean temperature and an improved clinical condition. In this trial no significant differences were found in clinical recovery between the two therapies as measured by group mean temperature, number of pigs requiring retreatment, and time to recovery. The conclusion that there was no important difference in clinical recovery between the treatment groups was made with a power of at least 90%.

Effects of ketoprofen and flunixin in pigs experimentally infected with Actinobacillus pleuropneumoniae.

The antipyretic effect of the non-steroidal anti-inflammatory drugs (NSAIDs) ketoprofen (3 mg/kg) and flunixin (2 mg/kg) were studied in pigs. The drugs were administered intramuscularly at 8 and 32 h following endobronchial challenge with Actinobacillus pleuropneumoniae. Infected (non-medicated) and non-infected (non-medicated) controls were used. Endobronchial challenge with Actinobacillus pleuropneumoniae induced laboured breathing, coughing, fever, reduced food and water consumption and increased white blood cell counts. At autopsy, pleuropneumonia was evident. Ketoprofen showed a highly significant antipyretic effect but flunixin did not. The decrease in food consumption of ketoprofen-treated pigs was significantly less than that of the infected (non-medicated) controls. Blood parameters were not significantly influenced by either NSAID and, at necropsy, gastric and renal side-effects were not observed for either drug.

Integrated quality control project: relationships between pathological findings detected at the slaughterhouse and information gathered in a veterinary health scheme at pig farms.

In September 1986 an Integrated Quality Control Project (IQC) was started in the Netherlands. Over a period of 2 years a veterinary herd health scheme was applied to 21 growing and finishing pig herds. Information was collected concerning medical treatment, classified as group treatment and individual treatment, clinical observations, and housing factors. The main purpose of this project was to investigate relationships between pathological findings detected at the slaughterhouse and information gathered on the farm. Medical group treatments, clinical observations and housing did not reliably predict pathological findings. Individual medical treatment for respiratory disorders, started in the last 30 days before slaughter, proved to be positively related to the pathological findings 'pneumonia' and 'pleuritis' detected at the slaughterhouse. This may be useful for selecting pigs at the slaughterhouse that will require special treatment for the removal of the pleura parietalis. During the first year of the project the average daily gain (ADG) was 27.2 g. more than during the preceding 2 years (P < 0.0001). Coughing was negatively related with the ADG. More than 75 coughs per compartment during 10 minutes, recorded more than 50 days before slaughter, was positively related to a depression of the ADG (20 g; P < 0.05). Relationships between group medical treatments and ADG were statistically significant, when the group medical treatment was started more than 20 days before slaughter. Negative influences on the ADG were higher if the treatment was started closer to the slaughter date.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of optimal age at first conception in gilts from data collected in commercial swine herds.

The purpose of this study was to find the economically optimal period of first conception in gilts, addressing the issues of lifetime reproductive performance and expected herd life. A profit equation was used to combine the effects into one economic parameter. The data were from 14,910 gilts on 54 farms throughout The Netherlands. The average number of pigs born alive in the first litter increased with older age at conception. In the second litter a similar, but much smaller, effect was observed. Age at first conception had no effect on number of pigs born alive in the third or greater litter. Gilts bred at an older age had a shorter expected herd life than gilts bred at a younger age. Evaluating the reasons for culling revealed that infertility became a more important reason with increasing age at first conception. The proportion culled for infertility increased linearly from 18% at conception on d 200 to 24.5% at conception on d 300. Combining the effect of litter size and herd life led to the conclusion that the profit per gilt (sow) was not significantly affected by her age at first conception. It is concluded that the optimal economic age at first conception was considered to be approximately 200 to 220 d of age when the cost of housing and feed of the gilt from entry to first conception were taken into account.

[Porcine Epidemic Abortion and Respiratory Syndrome (PEARS)-positive and PEARS-negative diagnoses in pig breeding and reproduction farms in The Netherlands using statistical methods]. / De diagnoses 'Porcine Epidemic Abortion and Respiratory Syndrome' (PEARS)-verdacht en PEARS-onverdacht op fok- en vermeerderingsbedrijven in Nederland met behulp van statistische methoden.

The diagnoses Porcine Epidemic Abortion and Respiratory Syndrome (PEARS)-positive and PEARS-negative can be made using the following data on herd performance: the average number of stillborn piglets per litter, the average number of live piglets per litter and the average piglet mortality before weaning. Depending on the number of litters used in calculating the averages, the extent to which two out of the three performance data should deviate to arrive at the diagnosis PEARS-positive is given in tables and figures. The same farm will only be diagnosed PEARS-negative if after some time at least two out of the three performance data deviate less than other criteria also provided in tables and figures.

Influence of rheumatoid synovial fluid and cells on proteoglycans in human cartilage explants. Modulation by piroxicam.

We have studied the effects of cell-free rheumatoid synovial fluid (RASF) and the conditioned medium (CM) from these cells on the proteoglycans (PGs) of normal human cartilage and the influence which piroxicam might have on these processes. Both RASF and the CM from RASF cells enhanced the PG release from the cartilage explants. The effects of the above mentioned fluids on the cartilage PG content depended on the metabolic state of the cartilage i.e. correlated inversely with the PG synthesis. Whether this was due to the presence of anabolic and catabolic factors in these fluids is discussed. Piroxicam had no adverse effect on the PGs of human cartilage in vitro. Piroxicam prevented the cartilage PG depletion when it was induced by the CM from RASF cells.

A longitudinal study of starvation in piglets and the introduction of a modified liver biopsy technique.

The effect of starvation on blood glucose, muscle glycogen and liver glycogen concentration was measured in a group of newborn piglets. Liver biopsies were obtained by using a modified version of the Menghini technique. No difference in length of survival time was observed between piglets that received water and those that did not. Piglets with higher birth weight survived longer. No relationship was found between initial liver glycogen content and survival time. Moreover, we concluded that plasma glucose levels are not reliable indicators of length of survival time. The interrelated changes in liver glycogen, muscle glycogen, and plasmaglucose concentrations found in this study correspond with those reported elsewhere. Moreover, the number of experimental animals needed for the study was markedly reduced. We conclude that the liver biopsy technique is valuable in longitudinal hypoglycaemia studies of piglets.

VAMPP: a veterinary automated management and production control program for swine breeding farms. I. Main features.

To improve the herd health and production management on swine breeding farms the Department of Herd Health and Ambulatory Clinic (University of Utrecht) developed a computer package. The basic package includes action lists, physical performance reports, analysis and economic programs. During the development of the software package it was emphasised that the programs should be user friendly (easy to handle, with no special codes), and should have an enormous flexibility to guarantee easy updating and a long life for the whole package. The Standard MUMPS system, which offers a unique data base structure and access method, was thus employed. The software runs on a central mini-computer with telephone lines for farmers and veterinary surgeons. In addition, the 'post-in post-out' method can be used; a micro-computer version of the package also is available.

VAMPP: a veterinary automated management and production control program for swine breeding farms. II. Output.

The Department of Herd Health and Ambulatory Clinic of the Veterinary Faculty (State University of Utrecht, The Netherlands) has developed the VAMPP package for swine breeding farms. The present article is the second of two articles describing the VAMPP-system. The objective of this computer package is to improve productivity on swine breeding farms through automated data processing and interpretation and action based on the computer output. The output produced by the VAMPP-programs is described in the present article and consists of: action lists, physical performance reports, analysis graphs and tables and economic reports.

The cycle index, an alternative to the farrowing index converted to a yearly basis.

The farrowing index is an important parameter for the evaluation of sow productivity. The value of the farrowing index calculated over a relatively short period of time and converted to a yearly basis, depends largely on the number of litters produced during this period. Random differences in the numbers of litters produced in subsequent periods will strongly influence the farrowing indices of these periods. The cycle index is designed to obtain a more valuable and consistent parameter. The cycle index is based on reproductive data of sows that farrowed during a certain period and the number of production days lost because of culling of sows during that period.