Jerome Cantor

Modulation of airspace enlargement in elastase-induced emphysema by intratracheal instillment of hyaluronidase and hyaluronic acid

The study examined how lung hyaluronic acid content influences airspace enlargement in elastase-induced emphysema. To determine the effect of a decrease in hyaluronic acid, hamsters received a single intratracheal instillment of hyaluronidase 24 h prior to administration of pancreatic elastase by the same route. One week later, these animals showed significantly greater airspace enlargement than controls sequentially instilled with saline and elastase (128 vs. 100 microns; p < .05). Conversely, intratracheal administration of hyaluronic acid immediately after elastase instillment resulted in a marked decrease in airspace enlargement at 1 week compared to controls receiving elastase followed by saline (82 vs. 122 microns; p = .005). Since hyaluronic acid has no elastase inhibitory capacity, its effect may involve extracellular matrix interactions not directly related to elastic fiber breakdown. This concept is supported by the finding that animals treated with hyaluronidase and elastase showed no greater loss of lung elastin than that observed in the saline/elastase control group, despite demonstrating a marked increase in airspace enlargement. Further work is needed to determine how hyaluronic acid influences airspace enlargement and to evaluate the potential use of this substance as a treatment for emphysema.

Attenuating effect of taurine on lipopolysaccharide-induced acute lung injury in hamsters

This study has evaluated the ability of the semiessential amino acid taurine to attenuate lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis in a small animal model. For this purpose, bacterial LPS (0.02mg in phosphate buffered saline (PBS) pH 7.4) was instilled intratracheally into female Golden Syrian hamsters, before or after a 3-day intraperitoneal treatment with a single dose (50mg/kg in PBS pH 7.4) of taurine. At 24h after the last treatment, lung tissue and bronchoalveolar lavage fluid (BALF) samples were collected. In comparison to samples from animals receiving only PBS pH 7.4, serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils in the BALF, and increased incidence of apoptosis, depletion of intracellular glutathione and evidence of inflammation confined to the parenchyma in the lung. In addition, LPS caused cells in the BALF to exhibit a higher expression of tumor necrosis factor-1, a higher activity of caspase-3, marked lipid peroxidation, and altered activities of catalase, glutathione peroxidase and superoxide dismutase relative to control samples. In contrast, a treatment with taurine was found to significantly attenuate all of the cellular and biochemical alterations induced by LPS, more so when given before rather than after the endotoxin. The present results suggest that taurine possesses intrinsic antiinflammatory and antioxidant properties that may be of benefit against the deleterious actions of LPS in the lung.

Elastin Degradation: An Effective Biomarker in COPD

Chronic obstructive pulmonary disease (COPD) is well recognized as one of the major health problems in the world (1,2). In the United States, it has moved up to the third leading cause of death (3). Th e disease has been defi ned as progressive and not fully reversible (2). Existing therapies off er benefi ts. Th erapies to reverse its progressive course have had limited success. Th ere is clear recognition that new and eff ective therapies are needed in this disease. New drug development is costly and fraught with failure so mechanisms must be sought to facilitate this development. Th e development of biomarkers of COPD is one such strategy. Th ere are several “defi nitions” of biomarkers. Th e US Food and Drug Administration operationalizes biomarkers as a reliable measure that can be used in at least one of three contexts to facilitate drug development: subject stratifi cation, dose ranging and as outcome measures (4). Th e National Institutes of Health (NIH) defi nes biomarkers as the “characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes or pharmacological responses to a therapeutic intervention”(5). Another criterion suggested for a biomarker that would be an outcome measure is (6) “a strong independent consistent association between the surrogate end point and the clinical endpoint” and “evidence from randomized control trials that improvements in the surrogate endpoint with a drug consistently leads to improvement in the target (clinical) endpoint.” At the present time, according to the FDA: “With the exception of lung function tests, there are no well validated biomarkers or surrogate endpoints that can be used to establish effi cacy for a drug for COPD”7 Th e following review is the fi rst in a series of brief reviews to be published in the Journal of COPD that will summarize potential biomarkers that may be used to facilitate development of novel treatments in COPD. Th is review brings together the evidence for matrix elastin degradation products as eff ective biomarkers for COPD. Elastin is synthesized in the extra-cellular space by elastin synthesizing cells which secrete the soluble precursor, monomer, tropoelastin, which is then cross linked mainly by two amino acids, desmosine and isodesmosine (Dl), which are derived by post-translational modifi cation of lysyl residues (8). Cross-linking transfers the soluble tropoelastin to the insoluble cross linked mature elastin fi ber. DI occur only in mature elastin and their presence in body fl uids is an indicator of degradation of mature elastic fi bers (9). Elastin occurs mainly in lung, blood vessels and skin. Elastin is present in bronchi and lung parenchyma. In the alveolar regions of the lung, elastin fi bers are deployed around alveolar ducts, the openings of alveoli and extensions into alveolar septa (10). Dissolution of elastic fi bers in the latter regions leads to tissue loss, altered alveolar structure and emphysema. In C O PD D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y Y al e D er m at ol og ic S ur ge ry o n 05 /1 8/ 15

Aerosolized recombinant human lysozyme ameliorates pseudomonas aeruginosa–induced pneumonia in hamsters

ABSTRACT As an alternative to conventional antibiotics, aerosolized recombinant human lysozyme (rhLZ) was used to treat experimentally induced pneumonia. Syrian hamsters were inoculated intratracheally with a nonmucoid strain of Pseudomonas aeruginosa (PA), then exposed to a 1.0%% solution of rhLZ in water for 2 hours per day for 3 consecutive days (controls were treated with aerosolized water alone). Compared to controls, the rhLZ-treated group showed statistically significant reductions in the following parameters: (1) lung histopathological changes, (2) bacterial colony-forming units in whole lung and bronchoalveolar lavage fluid (BALF), (3) total BALF leukocytes, (4) percent BALF neutrophils, and (5) alveolar septal apoptosis. Exposure to aerosolized rhLZ also resulted in a large increase in BALF lysozyme activity. These findings indicate that aerosolized rhLZ may be potentially useful in reducing the level of bacterial colonization and inflammation in the lungs of patients with PA pneumonia.

Aerosolized recombinant human lysozyme enhances the bactericidal effect of tobramycin in a hamster model of pseudomonas aeruginosa–induced pneumonia

ABSTRACT Previous studies from this laboratory have shown that aerosolized recombinant human lysozyme (rhLZ) mitigates Pseudomonas aeruginosa (PA)-induced pneumonia. In the current investigation, our laboratory tested the hypothesis that aerosolized rhLZ can potentiate the effects of tobramycin (TBMN), thereby reducing the effective dose of this agent in the treatment of PA-induced pneumonia. Syrian hamsters were instilled intratracheally with PA, then exposed to an aerosol containing either 1% rhLZ, 3 μg TBMN, or a combination of both agents. In contrast to the initial studies with rhLZ, which involved 3 separate aerosol exposures, only a single treatment was used in the current investigation. Twenty-four hours after completion of the aerosol regimen, the following parameters were measured: (1) whole-lung colony-forming units (CFU), (2) total bronchoalveolar lavage fluid (BALF) CFU, (3) lung histopathology, and (4) total BALF neutrophils. The combination of rhLZ and TBMN significantly reduced whole-lung and BALF CFU, as well as the inflammatory index, compared to TBMN alone. Similar results were seen in vitro with regard to bactericidal activity. These findings provide a rationale for clinical testing of rhLZ as an adjunct to commercial antibiotic treatment.

Preferential binding of lysozyme to elastic fibres in pulmonary emphysema

BACKGROUND Lysozyme is increased in inflammatory reactions and is a component of the extracellular matrix, but its possible role in lung diseases such as emphysema and interstitial fibrosis has not been investigated. METHODS To characterise differences in lysozyme content among normal, emphysematous, and fibrotic human lungs, tissue sections obtained from necropsy specimens were immunostained with rabbit polyclonal anti-human lysozyme antibody using the labelled streptavidin-biotin peroxidase method. The immunostained sections were evaluated semi-quantitatively (grading the degree of immunostaining on a scale of 0–4). To determine if degradation of the extracellular matrix affects lysozyme binding, hyaluronidase-treated normal lung tissues were incubated with egg white lysozyme, immunostained with the lysozyme antibody, which crossreacts with egg white lysozyme, and evaluated for degree of staining. RESULTS Lysozyme immunostaining was significantly increased in lungs with pulmonary emphysema compared with normal or fibrotic tissues (3.4 versus 1.6 and 1.9, respectively; p<0.05) and was preferentially associated with interstitial elastic fibres. Hyaluronidase-treated lung tissues incubated with lysozyme showed increased immunostaining for this protein compared with untreated controls (1.9 versus 1.2; p<0.05). CONCLUSIONS The results suggest that damage to elastic fibres and/or the surrounding extracellular matrix increases lysozyme binding. It is hypothesised that attachment of lysozyme to elastic fibres may interfere with their repair and possibly enhance the progression of pulmonary emphysema.

Short-Term Cigarette Smoke Exposure Predisposes the Lung to Secondary Injury

Brief exposure to cigarette smoke is not generally associated with pulmonary injury and may adversely affect the lung only if underlying disease is present. To test this hypothesis, our laboratory performed a series of experiments involving exposure of hamsters to second-hand cigarette smoke (2 h/day for 5 days), either immediately before or after induction of acute pulmonary injury by intratracheal administration of amiodarone. Compared to controls receiving amiodarone alone, hamsters pretreated with smoke showed significant increases in the following parameters: (1) lung inflammation graded on a scale of 0-4 (3.4 vs. 1.6; p < 0.001), (2) percentage of neutrophils in bronchoalveolar lavage fluid (BALF) (75.0 vs. 1.3; p < 0.001), (3) percentage of TNFR1-positive BALF macrophages (44.7 vs. 2.7; p < 0.001), and (4) apoptotic lung parenchymal cells per ten high-power microscopic fields (7.3 vs. 0.7; p < 0.001). Animals post-treated with smoke also showed significant increases in these parameters compared to controls but to a lesser degree than pre-exposed animals. With regard to human disease, such synergistic interactions may account for a significant portion of the morbidity associated with second-hand smoke exposure.

HJP272, A Novel Endothelin Receptor Antagonist, Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Hamsters

IntroductionPrevious studies from this laboratory indicate that endothelin-1 (ET-1), a potent vasoconstrictor, may play an important role in lipopolysaccharide (LPS)-induced release of neutrophils from the pulmonary microvasculature. To further test this concept, Syrian hamsters were treated with a novel endothelin receptor A (ETA) antagonist (HJP272) prior to intratracheal instillation of LPS.MethodsThe effect of HJP272 on the LPS-induced inflammatory reaction was determined by measuring: (1) lung histopathological changes, (2) total neutrophils in bronchoalveolar lavage fluid (BALF), (3) expression of tumor necrosis factor receptor 1 (TNFR1) by BALF macrophages, and (4) alveolar septal cell apoptosis.ResultsTreatment with HJP272 significantly reduced each of these parameters during a 24-hr period following LPS instillation, supporting the concept that limiting the activity of ET-1 may reduce the extent of lung injury. This hypothesis was further tested by giving ET-1 prior to LPS instillation, which resulted in a marked enhancement of LPS-induced lung inflammation, as measured by BALF neutrophils and TNFR1-positive macrophages. Furthermore, the increase in neutrophils resulting from treatment with ET-1 was significantly reduced by HJP272, again demonstrating the ability of ETA receptor antagonists to limit the influx of these cells into the lung.ConclusionsThese findings suggest a potential therapeutic role for these agents in diseases where neutrophils are a significant cause of lung injury, such as bronchopneumonia, respiratory distress syndrome, and chronic obstructive pulmonary disease.

The Ratio of Free to Bound Desmosine and Isodesmosine May Reflect Emphysematous Changes in COPD.

AbstractBackground The unique elastin crosslinks, desmosine and isodesmosine (DID) are significantly elevated in blood, urine, and sputum from patients with COPD, and may decline following treatment of the disease. However, the large degree of variance in this biomarker among COPD patients with similar levels of disease suggests that it has limited prognostic value with regard to the degree of lung disease in a given individual. As an alternative to measuring the total amount of DID, we propose using the ratio of free to peptide-bound DID, which may provide a better indication of overall lung disease.MethodsTo test this hypothesis, the free/bound DID ratio was measured in bronchoalveolar lavage fluid (BALF) from both hamsters with elastase-induced emphysema and controls not given the enzyme, using a combination of liquid chromatography and tandem mass spectroscopy. This ratio was then correlated with airspace enlargement, as measured by the mean percentage of lung surface area at ×100 microscopic magnification.ResultsThere was a significant negative correlation between the free/bound DID ratio in BALF and lung surface area. However, there was no correlation between this ratio and total BALF DID, suggesting that free/bound DID is unrelated to the immediate rate of breakdown of elastic fibers, and may instead measure the cumulative effect of elastase injury in the lung.ConclusionsThe free/bound DID ratio may be a useful measure of emphysematous changes in the lung and might also serve as a screening procedure for healthy smokers and other individuals at risk for developing COPD.

What percolation theory can tell us about COPD

Damage to the lung elastic fiber network is largely responsible for the distention and rupture of alveolar walls in chronic obstructive pulmonary disease (COPD). It has therefore been suggested that blood or urine levels of the unique elastic fiber crosslinks, desmosine and isodesmosine (DID), may serve as a biomarker for the progression of the disease. The prognostic value of DID may be limited, however, by the large degree of variance associated with their measurement in patients with COPD. To overcome this problem, we propose that specific patterns of DID release from damaged elastic fibers, rather than their absolute quantity, may provide a better indication of morphological changes in the lungs of patients with COPD. Using percolation theory to model the elastic fiber network in the lung, it will be shown that the relative amounts of damaged and intact elastic fibers may be reflected at the molecular level by urinary levels of free and peptide-bound DID, respectively. The self-similar nature of percolation networks further suggests that detachment of crosslinks from elastic fibers may be analogous to the rupture of alveolar walls in COPD. Consequently, the ratio of free to bound DID may be a measure of emphysematous changes in this disease.