MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

Potential Use of Surface-active Agents for Controlling Mycoplasma Contamination in Animal Cell Cultures

Seven nonionic detergents, which were determined to be relatively nontoxic to selected animal cell cultures, were tested for their lethal effect on the GDL strain of Mycoplasma hyorhinis. Of the seven detergents tested, five were found to cause complete lysis of the organism in vitro within 24 hr at 37 C. These detergents included Triton WR-1339 and Tweens 20, 40, 60, and 80. When different concentrations of the detergents were tested, Tween 80 was found to be the most effective and Triton WR-1339 the least effective in lysing the mycoplasmata. These same five detergents were used to treat a rat nephroma cell line which was chronically infected with the GDL strain. The mycoplasmata were eliminated from those cultures treated with Triton but they persisted in cultures exposed to the Tween compounds. The Triton-treated cells remained free from infection over a 7-month period, as determined both by cultural methods and fluorescent-antibody staining. The "cure" was effected by treating the cells for either 48 hr with maintenance media containing 1 mg of Triton per ml or for 96 hr with a concentration of 500 mug/ml. Triton was also effective in eliminating the GDL, strain from experimentally infected rat embryo cells after a 48-hr treatment with a concentration of 1 mg/ml. Four other species of Mycoplasma, which were completely lysed by Triton in vitro, were not eliminated from experimentally infected cells by a single treatment with Triton, although the severity of the infection was apparently reduced.     

Purification of the Kilham Rat Virus

A purification procedure is described for the isolation of Kilham rat virus (RV) from infected suckling hamster kidney and liver suspensions. The procedure involved a combination of sonic treatment, differential centrifugation, butanol-chloroform extraction, agar column flow diffusion, and potassium tartrate density gradient centrifugation. The purified virus retained its infectivity and was specifically neutralized by RV hyperimmune antiserum. Electron micrographs from the RV band (density 1.31 g/ml) showed numerous homogeneous particles approximately 22 mmu in diameter.     

In vitro suppression of the phagocytic response of fish macrophages by tetracyclines

Tetracycline (TC) and oxytetracycline (OTC) caused a dose-dependent suppression of the chemiluminescence (CL) emitted by phagocytes from the kidney of rainbow trout, Salmo gairdneri , using zymosan or latex beads as the stimulus. Compared to the control response without antibiotics, partial but significant suppression was found after exposing the cells to OTC concentrations of 0.1–50.0 μg ml⁻¹. Cells exposed to 100 or 500 μg ml⁻¹ OTC showed CL responses below the base levels elicited by control cells. Comparable results were obtained with cells exposed to TC and stimulated by the same stimuli. The kinetics of the CL response and the suppressive effects of the antibiotics were similar in cells from individual fish but the magnitude of responses varied. No acclimation occurred following extended exposure to the drugs.     

Effects of beta-blockade on exercise capacity of trained and untrained men: a hemodynamic comparison

To study the effects of cardiovascular fitness on hemodynamic responses to exercise during beta-adrenergic blockade (BAB), submaximal [60% of maximum O2 uptake (VO2max)] and maximal treadmill exercise data were collected in 11 trained (T, VO2max 63.3 ml X kg-1 X min-1, 26.8 yr) and 11 untrained (UT, VO2max 44.5 ml X kg-1 X min-1, 25.0 yr) male subjects. Subjects completed two maximal control tests followed by a randomized, double-blind series of maximal tests after 1-wk treatments with placebo (PLAC), propranolol (PROP, 160 mg/day, beta 1- and beta 2-blockade), and atenolol (ATEN, 100 mg/day, beta 1-blockade). Treatments were separated by 1-wk washout periods. At 60% of control VO2max T and UT subjects experienced no reductions in O2 uptake (VO2) with either drug. Submaximal heart rate (HR, beats/min) was 134.8 PLAC, 107.0 PROP, 107.9 ATEN (P less than 0.05 both drugs vs. PLAC) in T subjects and 141.1 PLAC, 106.1 PROP, and 105.0 ATEN (P less than 0.05 both drugs vs. PLAC) in UT subjects. Cardiac output (1/min) for T was 17.3 PLAC, 16.9 PROP, 16.5 ATEN (P less than 0.05 ATEN vs. PLAC in T only) and for UT it was 12.2 (PLAC), 11.7 (PROP), 11.5 (ATEN) (P less than 0.05 both drugs vs. PLAC in UT). Stroke volume increased from 129.8 ml (PLAC) to 158.6 (PROP) and 156.2 (ATEN) in T (P less than 0.05 both drugs vs. PLAC) and from 86.8 (PLAC) to 110.0 (PROP) and 109.8 (ATEN) (P less than 0.05 both drugs vs. PLAC) in UT. The increases in stroke volume (SV) were similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein

Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an approximately 72 amino acid consensus motif and the first four of these domains, denoted WLN1-4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5-6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5-6, and 6, denoted by WLN1, WLN3, WLN4, WLN5-6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.     

Copy number variation in familial Parkinson disease

Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Uncoupling of proopiomelanocortin (POMC) fragments is related to self-injury

Proopiomelanocortin (POMC) contains several interesting, behaviorally active peptides. Release patterns of these fragments have been related to bizarre episodes of self-injurious behavior (SIB) among autistic individuals. Moreover, elevation in beta-endorphin (betaE) but not ACTH levels was associated with a positive response to an acutely administered, centrally acting opioid blocker among autistic individuals exhibiting SIB. In the present study, POMC fragments were measured in 12 self-injurious patients before and after long term (3 month) treatment with an opiate blocker naltrexone (NTX). POMC fragments were sampled from blood collected at the beginning of the baseline and placebo-controlled treatment phases of the study. Results indicated that the co-release (coupling) of POMC fragments were stable over time and the profile of POMC fragments in plasma predicted the effectiveness of a CNS acting drug in autistic subjects who self-injure.