Clinical practice variation in acute severe burn injury.

The New South Wales (NSW) Statewide Burn Injury Service Database was reviewed to identify variations in clinical practice with respect to care of severely burn-injured patients in intensive care. We compared differences in practice relating to duration of endotracheal intubation and surgical grafting. In this retrospective observational study, we reviewed all intensive care unit (ICU) admissions to the two NSW adult burns centres, ICU A and ICU B, between January 2008 and December 2015. Data were analysed for association between duration of intubation and outcome. There were 855 admissions to adult ICU, with a significant difference in the percentage total body surface area (% TBSA) of burn and inhalation injury between patients in the two units. There was a significant difference in duration of intubation and ICU length of stay (LOS) between the units, which persisted when adjusted for age, % TBSA and inhalational injury. When analysing patients with more severe burns (>20% TBSA or intubated), the difference in duration of intubation remained significant (median of three days [interquartile range, IQR, 1-11 days] in A and 2 days [IQR 1-6 days] in B, P=0.003) as did ICU LOS (median 3 days [IQR 2-11 days] for A and 2 days [IQR 1-6 days] for B, P <0.0005). There was no significant difference in mortality between the two units for the severe or the more severe subgroup of burns when adjusted for age, % TBSA and inhalational injury (adjusted odds ratio, OR, for mortality 1.17 [95% confidence intervals 0.6 to 2.3, P=0.65]). There were significant differences in clinical practice, including duration of intubation, between the two ICUs. Longer intubation was associated with a longer ICU LOS, but was not associated with a difference in mortality. Large collaborative, prospective multicentre studies in severe burns are needed to identify best practice and variations in practice to determine if they are associated with increased mortality and/or cost.

Evidence-based evolution of the high stakes postgraduate intensive care examination in Australia and New Zealand.

The fellowship examination for intensive care medicine in Australia and New Zealand, first held in 1979, has undergone four major periods of development and change since inception. These periods are characterised as: 1. 1979 to 1996--initiation and establishment of the exam as a relevant and comprehensive assessment process for a new specialty. 2. 1997 to 2001--revision to increase breadth of coverage, increase reliability for a growing number of candidates and ensure that each candidate received the same exam: Expansion: to incorporate assessment of CanMEDS skills (including communication, procedures and professional qualities). Lengthening: to increase the number of exposures, to ensure reliability. Quarantining of candidates: to allow the provision of a similar exam for each candidate. 3. 2002 to 2006--increasing emphasis on examiner training, standard setting and increasing feedback to candidates to improve the educational experience and guide exam preparation. Blueprinting of questions to maintain validity. 4. 2008 onwards--logistic revision to ensure feasibility for a rapidly growing number of candidates and refinement to apply modem standard setting and quality control. The exam has been regarded as a 'tough but fair' assessment in its 30 years of existence and the committee overseeing its development has aimed to continually review the process to maintain those qualities as well as reliability, validity and feasibility. The increasing number of candidates has allowed accumulation of usable statistics but has tested the feasibility of running such a labour intensive exam. To date, there have been 800 presentations to the exam with 498 successful candidates.

The effects of salivary gland extracts from Boophilus microplus ticks on mitogen-stimulated bovine lymphocytes.

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% +/- 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% +/- 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 microg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.

A comparison of the immunosuppressive effects of salivary gland extracts from two laboratory strains of Boophilus microplus.

This study addresses three questions related to the immune response of cattle to tick salivary gland extracts. Firstly, is there a difference in the inhibition of proliferation of Concanavalin A (ConA) stimulated bovine lymphocytes induced by salivary gland extracts of the N and Y strains of Boophilus microplus? Second, is there a difference in the development rate of the Y and N tick strains? Third, does the host affect the inhibitory effect of salivary gland extract on the proliferation of ConA stimulated lymphocytes from the two tick strains? Salivary gland extract of the Y strain inhibited in vitro proliferation of lymphocytes stimulated by ConA significantly more than that of the N strain, when each strain was raised on different animals. A difference in the development rate was observed between the tick strains when raised on the same animal, with female ticks of the Y strain developing faster and reaching a greater fully engorged weight than ticks of the N strain. The difference in their rate of development did not appear to contribute to a difference in inhibitory effects of the salivary gland extracts and there was no difference between the inhibitory effects of salivary gland extracts from both strains. However, when Y strain ticks were raised on different animals, there was a significant difference in the inhibition of lymphocyte proliferation between the two salivary gland extracts. Therefore, it was concluded that there is no difference between the inhibitory effects of the two tick strains and that the host has an influence on salivary gland extract composition of B. microplus and its inhibitive properties.

Early invasive revascularisation for patients critically ill after acute myocardial infarction: impact on outcome and ICU resource utilisation.

OBJECTIVE: To assess the value of early invasive revascularization for the initial management of critically ill patients after acute myocardial infarction in the daily practice of a University-affiliated referral hospital and to gauge the impact of such a strategy on the intensive care unit. PATIENTS AND METHODS: A prospective observational study on all patients admitted to the Royal North Shore hospital who had acute pulmonary oedema and/or shock prior to acute angiography for acute myocardial infarction from January 1(st), 1998 to December 31, 2001. RESULTS: During the study period 846 patients with acute myocardial infarction had coronary artery angiography, 139 had acute pulmonary oedema and/or shock prior to angiography. The average age was 70 years, 65% of whom were male. Approximately 70% of these patients were admitted to the intensive care unit and coronary artery bypass surgery was performed on 38%. Of those patients admitted to the intensive care unit, 95% required mechanical ventilation, 81% required inotropic support and 50% required intra-aortic Balloon counterpulsation. In-hospital mortality was 32%, 6 weeks mortality was 38% and 6 month mortality was 42%. CONCLUSIONS: Our results confirm the benefit of early invasive revascularisation for critically ill patients after acute myocardial infarction although a substantial amount of intensive care unit resources and cardiothoracic surgical expertise were required.

Pathological and immunological changes after challenge infection with Pasteurella multocida in naive and immunized calves.

Challenge infections of calves with Pasteurella multocida were established to characterize the local inflammatory response and determine the effect of previous exposure to live bacteria on the post-challenge immune response. Experimental infections were established by intratracheal inoculation of P. multocida in both naive calves and calves that had been previously vaccinated with two subcutaneous (s.c.) injections of live bacteria. Histological, immunohistological and cytokine expression studies were performed on bronchoalveolar lavage (BAL) samples, lung parenchymal tissues and lung lymph nodes (LN). In comparison to uninfected control animals in which no lung lesions were observed, a patchy to confluent bronchopneumonia was observed following infection of naive calves, characterized by abscess formation, haemorrhage, oedema and suppurative consolidation. Cellular analysis following infection of naive animals was characterized by an influx of neutrophils in the BAL, with macrophages and dendritic cells observed in the lesion perimeter. A significant increase in the number of CD8(+) blasts expressing MHC (major histocompatibility) II was also observed in the BAL of infected calves. Decreased expression of interleukin (IL)-1 beta and increased expression of IL-8 compared to naive unchallenged controls was apparent in lung LN. In comparison, a more limited pathology was observed in vaccinated animals post-challenge, indicating partial protection conferred by the s.c. immunization with live bacteria. Studies of vaccinated animals showed the presence of bronchial-associated lymphoid tissue (BALT) in the lung tissue and an increase in the number of B-cells and CD4(+) T-cells expressing MHCII in the lung LN after challenge. In contrast to primary infection, there was no significant influx of neutrophils in the BAL. Instead, a population of newly recruited monocytes/macrophages was observed. Increased IL-2 expression and decreased IL-8 expression was observed in the LN, while IL-1 beta expression was not detected. The reduced neutrophil and increase monocyte response in the vaccinated calves may be associated with significant changes in the gamma delta T lymphocyte population in the BAL.

The protective effect of niacinamide on ischemia-reperfusion-induced liver injury.

Reperfusion of ischemic liver results in the generation of oxygen radicals, nitric oxide (NO) and their reaction product peroxynitrite, all of which may cause strand breaks in DNA, which activate the nuclear enzyme poly(ADP ribose)synthase (PARS). This results in rapid depletion of intracellular nicotinamide adenine dinucleotide and adenosine 5'-triphosphate (ATP) and eventually induces irreversible cytotoxicity. In this study, we demonstrated that niacinamide, a PARS inhibitor, attenuated ischemia/reperfusion (I/R)-induced liver injury. Ischemia was induced by clamping the common hepatic artery and portal vein of rats for 40 min. Thereafter, flow was restored and the liver was reperfused for 90 min. Blood samples collected prior to I and after R were analyzed for methyl guanidine (MG), NO, tumor necrosis factor (TNF-alpha) and ATP. Blood levels of aspartate transferase (AST), alanine transferase (ALT) and lactate dehydrogenase (LDH) which served as indexes of liver injury were measured. This protocol resulted in elevation of the blood NO level (p < 0.01). Inflammation was apparent, as TNF-alpha and MG levels were significantly increased (p < 0.05 and p < 0.001). AST, ALT and LDH were elevated 4- to 5-fold (p < 0.001), while ATP was significantly diminished (p < 0.01). After administration of niacinamide (10 mM), liver injury was significantly attenuated, while blood ATP content was reversed. In addition, MG, TNF-alpha and NO release was attenuated. These results indicate that niacinamide, presumably by acting with multiple functions, exerts potent anti-inflammatory effects in I/R-induced liver injury.

The lung is the major site that produces nitric oxide to induce acute pulmonary oedema in endotoxin shock.

1. The present study was undertaken to determine the locus of nitric oxide (NO) production that is toxic to the lung and produces acute pulmonary oedema in endotoxin shock, to examine and compare the effects of changes in lung perfusate on endotoxin-induced pulmonary oedema (EPE) and to evaluate the involvement of constitutive and inducible NO synthase (cNOS and iNOS, respectively). 2. Experiments were designed to induce septic shock in anaesthetized rats with the administration of Escherichia coli lipopolysaccharide (LPS). Exhaled NO, lung weight (LW)/bodyweight (BW) ratio, LW gain (LWG) and lung histology were measured and observed to determine the degree of EPE 4 h following LPS. The EPE was compared between groups in which LPS had been injected either into the systemic circulation or into the isolated perfused lung. The lung perfusate was altered from whole blood to physiological saline solution (PSS) with 6% albumin to test whether different lung perfusions affected EPE. Pretreatment with various NOS inhibitors was undertaken 10 min before LPS to investigate the contribution of cNOS and iNOS to the observed effects. 3. Endotoxin caused profound systemic hypotension, but little change in pulmonary arterial pressure. The extent of EPE was not different between that induced by systemic injection and that following administration to isolated lungs preparations. Replacement of whole blood with PSS greatly attenuated (P < 0.05) EPE. In blood-perfused lungs, pretreatment with NOS inhibitors, such as Nomega-nitro-L-arginine methyl ester, aminoguanidine and dexamethasone, significantly prevented EPE (P < 0.05). 4. The major site of NO production through the whole blood is in the lung. The NO production mediated by the iNOS system is toxic to the endothelium in the pulmonary microvasculature. Inhalation of NO for patients with sepsis may be used with clinical caution. Therapeutic consideration of lung extracorporeal perfusion with PSS and pharmacological pretreatment with iNOS inhibitors may be warranted.

Involvement of CYP2E1 and carboxylesterase enzymes in vinyl carbamate metabolism in human lung microsomes.

Previous studies have shown that CYP2E1 and carboxylesterase enzymes contributed to vinyl carbamate (VC) metabolism in murine lung. Moreover, these studies have implicated CYP2E1 and the carboxylesterases in bioactivation and detoxication, respectively. Here we have tested the hypothesis that CYP2E1 and carboxylesterase enzymes are involved also in VC metabolism in human lung. Demethylation of N-nitrosodimethylamine (NDMA) is an enzyme activity associated with CYP2E1, and was used as a catalytic marker for this P450 in human lung microsomes. NDMA demethylase activity in lung microsomes from 10 patients ranged from 36.9 +/- 1.0 to 82.4 +/- 2.4 pmol/mg protein/min. Significant decreases (40-65%) in demethylase activity were detected in lung microsomes incubated with VC and NADPH, compared with the controls in which incubations were performed with only VC or only NADPH. Preincubation with the CYP2E1 inhibitor diallyl sulfone also significantly decreased demethylase activity, and abrogated the VC-induced effect. Similarly, preincubation of lung microsomes with a human CYP2E1 inhibitory monoclonal antibody ameliorated the VC-induced reduction in demethylase activity. Microsomal carboxylesterase activity in lung microsomes from 10 patients ranged from 19.02 +/- 2.28 to 48.18 +/- 4.34 nmol/mg protein/min, and was significantly decreased (25-45%) in microsomes incubated with phenylmethylsulfonyl fluoride, an inhibitor of the carboxylesterase enzyme. Preincubation of lung microsomes with phenylmethylsulfonyl fluoride and subsequent incubation with VC and NADPH exacerbated the reduction (60-80%) in demethylase activity evoked by reaction with VC and NADPH. These results are consistent with a role for the CYP2E1 enzyme and microsomal carboxylesterases in VC metabolism.

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis.

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.

Opposition of rapid baroreceptor resetting by prostanoids in rabbits.

Arterial baroreceptors reset rapidly within minutes during acute hypertension; baroreceptor pressure threshold (Pth) is increased and the pressure-baroreceptor activity relation is shifted to the right. The purpose of the present study was to determine if prostacyclin (PGI2) or other prostanoids, released during acute hypertension modulate the magnitude of baroreceptor resetting. Baroreceptor activity was recorded from the vascularly-isolated carotid sinus during distension of the sinus with slow pressure ramp in rabbits anesthetized with chloralose. Pressure-activity curves were generated after holding carotid sinus pressure for 10-15 min from 30 to 100 mmHg. In control, the elevation of holding pressure increased Pth from 44+/- to 65+/-5 mmHg (p < 0.05, n = 12). In the presence of PGI2 (20 microM), Pth averaged 43+/-4 and 45+/-3 mmHg (n = 12) after holding pressure at 30 and 100 mmHg, respectively. In the control group before exposing the carotid sinus to indomethacin, an elevation of holding pressure increased Pth from 49+/-2 to 71+/-3 mmHg (p < 0.05, n = 12). After inhibition of the endogenous formation of prostanoids with indomethacin (20 microM), Pth increased by a significantly greater extent from 61+/-2 to 90+/-3 mmHg (p < 0.05, n = 12) with the increase in holding pressure. The slope of the pressure-activity curve (baroreceptor gain) was not influenced by the change in holding pressure. It was increased significantly by PGI2, while decreased by indomethacin. Neither the change in holding pressure nor PGI2 affected the circumferential wall strain of carotid sinus over a wide range of pressure alteration. The results suggest that PGI2 or other prostanoids released during acute hypertension sensitizes baroreceptors and provides a negative feedback mechanism that opposes and limits the magnitude of rapid baroreceptor resetting.

Putative salivary allergens of the cat flea, Ctenocephalides felis felis.

The cat flea, Ctenocephalides felis felis, is the major initiator of flea bite hypersensitivity in dogs. Previous analyses of whole extracts of the flea and flea salivary secretions have failed to identify the allergens responsible. We dissected >2000 salivary glands from adult female fleas, extracted them into buffered saline containing protease inhibitors and fractionated the extract using gel permeation HPLC. Dogs were classified as hypersensitive to fleas (flea-feeding positive, FF+) or insensitive (flea-feeding negative, FF-) using a provocative test with live fleas. The allergenicity of the components of the salivary gland extract was tested by intradermal injection of samples of the column eluates. Dogs were also injected intradermally with a sample of whole salivary gland extract, and with histamine as a positive control. Negative control injections consisted of eluate from the column collected prior to fractions containing any protein. The skin of FF- dogs either did not respond or had a minimal response (a bleb approximately 2 mm larger than the injection blebs at the negative control injection sites) to all fractions and to the whole extract; histamine control injections produced positive responses (defined as wheals 5 mm greater than the blebs at the negative control injection sites) in all dogs. The skin of three of the nine FF+ dogs reacted positively to injection of a fraction containing protein/s with apparent MW 40k. Five other FF+ dogs reacted positively to the fractions containing proteins with apparent MW 12-8k. A single dog responded with very large, red wheals to injection of both the approximately MW 40k and MW12-8k fractions. These findings suggest that proteins with apparent MW 40k and MW 12k-8k are important in flea bite hypersensitivity. This work also supports a previous finding that mice which had been exposed to flea bites had antibodies to proteins with approximately MW 40k that were detected in salivary secretions of the flea.

Anti-immunoglobulin antisera used in an ELISA to detect antibodies in barramundi Lates calcarifer to Cryptocaryon irritans.

Immunoglobulins (Ig) in serum from barramundi vaccinated with bovine serum albumin (BSA) were purified by ammonium sulphate precipitation and affinity chromatography using BSA as the ligand. The BSA-binding activity of eluted putative Ig fractions was assessed by enzyme-linked immunosorbent assay (ELISA) before being pooled and characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Double affinity purification did not improve the purity of the Ig preparation compared to single affinity purification. Barramundi Ig were injected into sheep to produce anti-Ig antisera which were assessed in an indirect ELISA as the secondary antibody to detect serum Ig in barramundi vaccinated with Cryptocaryon irritans theronts. Affinity-purified Ig induced a more specific reagent for use as secondary antibody in ELISA than did normal whole-barramundi sera. The heavy (H) chain of barramundi Ig had an apparent molecular weight of 70 kDa while that of the light (L) chain was 27 kDa in SDS-PAGE studies. Under non-reducing conditions 2 putative populations of Ig were identified, at 768 and 210 kDa. The N-terminal sequence of the barramundi Ig H chain showed 78% homology with channel catfish Ictalurus punctatus Ig H chain sequence.

Detection and measurement of canine alpha-macroglobulins by enzyme immuno-assay.

The alpha-macroglobulins are broad-specificity protease inhibitors important in the regulation and clearance from circulation of biologically active proteases. Inappropriate protease activation may be a feature of canine acute pancreatitis and the ability of the animal to clear these proteases may be important in determining survival. An enzyme immunoassay for the detection and measurement of canine alpha-macroglobulins in plasma samples was developed. A reference range for the canine alpha-macroglobulins of 1.20-2.72 mg ml-1 was established from a panel of canine plasma samples, and the stability of the alpha-macroglobulins in plasma samples stored at 4 degrees C was investigated. Changes in the level of the alpha-macroglobulins during disease states involving increased endogenous protease activity can now be investigated using a rapid, repeatable and quantifiable assay.

Inactivation of cytochrome P-450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes by vinyl carbamate in murine pulmonary microsomes.

We tested the hypothesis that vinyl carbamate (VC) is metabolized in vitro by cytochrome P-450 and carboxylesterase enzymes in murine lung. Incubations with VC and an NADPH-generating system produced a 50% decrease in N-nitrosodimethylamine (NDMA) demethylation and a corresponding loss in the amounts of immunodetectable CYP2E1. Preincubation of microsomes with a CYP2E1 inhibitory antibody or the CYP2E1-selective inhibitor diallyl sulfone (DASO2) inhibited demethylase activity; no alterations were detected upon subsequent exposure to VC. Carboxylesterase-mediated hydrolysis of p-nitrophenyl acetate was reduced by 22% in microsomes incubated with VC. Decreased carboxylesterase activity also was detected in microsomes incubated with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of hydrolase A, a carboxylesterase isozyme. No change in enzyme activity was detected when microsomes were subsequently incubated with VC. The loss in carboxylesterase activity correlated with decreased immunodetectable hydrolase A in microsomes incubated with VC, PMSF, or PMSF and VC. The reduction in VC-induced NDMA demethylase activity was increased to 85% of the control in microsomes previously incubated with PMSF, and this corresponded with a marked decrease in CYP2E1 immunoreactivity in the immunoblots. Covalent binding of VC to proteins was detected in microsomes incubated with VC and an NADPH-generating system. Binding was inhibited in microsomes preincubated with either an inhibitory CYP2E1 antibody or DASO2. In contrast, binding levels were augmented in microsomes preincubated with PMSF. These data supported VC metabolism by CYP2E1 and hydrolase A in murine lung microsomes and is consistent with involvement of CYP2E1 and hydrolase A in the activation and detoxication of VC, respectively.

Sustained-release delivery systems and their application for endoparasite control in animals.

A solid formulation of a potent anthelmintic macrocyclic lactone, moxidectin, was administered using a non-degradable delivery device to discharge the agent into the subcutaneous tissues of sheep. In vivo release was monitored in sheep indirectly using faecal egg counts. Using a dose of 0.2 mg moxidectin/kg body weight when applied in the form of a solid pellet, protection of sheep against Haemonchus contortus challenge was conferred to a level greater than that of sheep which received Cydectin, the commercial liquid injectable form delivered at the same dosage. The anthelmintic efficacy of the solid formulation was assessed at four dosage levels in sheep and it was demonstrated that the dosage of anthelmintic agent could be reduced to 1/6 of the present recommended injectable dose. When two pellets containing the recommended dose of moxidectin were loaded into a non-degradable delivery device, the period of H. contortus control was extended from 42 to 183 days. Antibody levels of sheep receiving repeated infections of H. contortus L3 larvae and treated with moxidectin-loaded devices were reduced significantly compared to the levels observed in sheep treated with Cydectin (p < 0.0005). This implies that the group treated with the moxidectin-loaded devices was exposed to a reduced antigenic load compared to sheep treated with placebo devices, and sheep treated with Cydectin. The antibody levels generated in the sheep treated with placebo devices were no different to those treated with Cydectin. Application of this sustained release device may allow the control of nematode diseases in livestock throughout an entire season with a single administration.

Isozyme-selective metabolism of ethyl carbamate by cytochrome P450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes in murine liver microsomes.

Cytochrome P450 and carboxylesterase enzymes have been implicated in the metabolism of the carcinogen ethyl carbamate (EC). In this study, we have used a murine liver microsomal system to investigate the relative contributions of P450 and carboxylesterase isozymes to hepatic metabolism of EC. N-Nitrosodimethylamine (NDMA) demethylation and p-nitrophenyl acetate (PNA) hydrolysis were used as catalytic markers of CYP2E1 and carboxylesterase enzymes, respectively. Incubation of liver microsomes with EC (1 mM) produced slight but significant decreases in NDMA demethylation and PNA hydrolysis activities. Incubation of microsomes with paraoxon (PAX), a general carboxylesterase inhibitor, or phenylmethylsulfonyl fluoride (PMSF), a specific inhibitor of hydrolase A, produced decreases of 85 and 45%, respectively, in carboxylesterase activities; neither of the inhibitors elicited alterations in levels of NDMA demethylation. Reaction of microsomes with either PAX or PMSF and then with EC exacerbated the reduction (285%) of NDMA demethylation, and this loss corresponded to decreases in immunodetectable CYP2E1 content. The reduction in PNA hydrolysis activity induced by PAX, PMSF, or EC correlated with decreased immunodetectable hydrolase A in liver microsomes; however, reaction with PAX and not PMSF or EC resulted in loss of immunoreactivity for hydrolase B. These data correlated with levels of covalent binding of [ethyl-14C]EC to liver microsomes, which were significantly elevated in incubations conducted with PAX or PMSF. Antibody inhibition of the CYP2E1 enzyme significantly reduced levels of binding to microsomal proteins, compared with control levels. These results are consistent with the premise that EC is metabolized by CYP2E1 and hydrolase A in liver microsomes of mice.

Metabolism of ethyl carbamate by pulmonary cytochrome P450 and carboxylesterase isozymes: involvement of CYP2E1 and hydrolase A.

The lung is highly susceptible to ethyl carbamate (EC)-induced tumorigenesis. Our goal in this study was to investigate the in vitro isozyme-selective metabolism of EC in lung microsomes by cytochrome P450 and carboxylesterase enzymes. Our results showed that incubations with EC produced significant reduction in p-nitrophenol (PNP) hydroxylation and N-nitrosodimethylamine (NDMA) demethylation; there were no alterations in 7-pentoxyresorufin- and 7-ethoxyresorufin O-dealkylase activities. Reaction of microsomes with an inhibitory CYP2E1 antibody and subsequent reaction with EC abolished the EC-induced diminution in NDMA demethylase activity. Carboxylesterase activity, as assessed by hydrolysis of p-nitrophenyl acetate, was significantly decreased in microsomes incubated with EC. Reactions with EC in conjunction with the carboxylesterase inhibitors, paraoxon (PAX) or phenylmethylsulfonyl fluoride (PMSF), abolished the EC-induced decrease in carboxylesterase activity; PAX is a broad-spectrum carboxylesterase inhibitor, whereas PMSF is a specific inhibitor of hydrolase A, a carboxylesterase isozyme. Incubations of EC in combination with either PAX or PMSF exacerbated the EC-induced reduction in PNP hydroxylase and NDMA demethylase activities. Alterations in immunodetectable CYP2E1 protein levels were not apparent in microsomes incubated with EC alone, but the amounts were decreased in reactions with EC in conjunction with either PAX or PMSF. Immunoblotting with antibodies for the carboxylesterase isozymes, hydrolase A and B, revealed loss of immunodetectable hydrolase A in microsomes incubated with EC, PAX, or PMSF. However, immunodetectable hydrolase B was only decreased in microsomes reacted with PAX but not with PMSF or EC. These findings correlated with our covalent binding data, which showed that levels of binding of [14C-ethyl]EC to lung microsomes were significantly higher in incubations conducted in conjunction with PAX or PMSF, compared with control levels. Antibody inhibition of the CYP2E1 enzyme significantly reduced the extent of binding. Our results demonstrated that EC metabolism in lung microsomes, as estimated from magnitudes of covalent binding, is mediated by the P450 isozyme CYP2E1 and the carboxylesterase isozyme hydrolase A.

Characterization of cytokine profiles and double-positive lymphocyte subpopulations in normal bovine lungs.

OBJECTIVE: To characterize cytokine profiles and lymphocyte subpopulations in lung parenchyma and bronchoalveolar lavage (BAL) fluid from normal bovine lungs. ANIMALS: Eight 12- to 18-month-old cattle. PROCEDURE: Cell populations in BAL fluid and collagenase-digested lung parenchyma were analyzed by flow cytometry and monoclonal antibodies. Proportions of total cell populations were determined, using Giemsa-stained cytospots. Distribution of lymphocytes within the lung parenchyma was analyzed by immunohistochemistry, and cytokine mRNA species in the parenchyma were characterized by use of reverse transcriptase-polymerase chain reaction analysis. RESULTS: Cytokine profiles indicated high amounts of mRNA for interleukins 6 and 10 and transforming growth factor beta. In the BAL fluid and lung parenchyma, macrophages were the predominant cell type, although the proportion was lower in the parenchyma. Lymphocytes made up approximately 3% of both cell populations. Common to both lung compartments was the predominance of CD2+ and gamma delta T cells over B lymphocytes. There were more CD8+ T cells than CD4+ T cells in both compartments. The gamma delta cells made up approximately 9% of the lymphocyte populations. Two-color flow cytometry revealed CD8+ gamma delta T cell and CD8+CD5- populations that were unique to BAL fluid. In the BAL fluid and parenchyma, most CD4+ and CD8+ T cells expressed high amounts of CD44, a characteristic of memory T cells. The gamma delta T cells were CD44(10), as were B cells in the lung parenchyma. The B cells from BAL fluid expressed high amounts of CD44. Immunohistologic analysis of lung tissue revealed bronchus-associated lymphoid tissue structures with distinctive germinal center organization of B cells encompassed by CD4+ T cells. CONCLUSIONS: Results provided normal values for comparison with those of other species and with the bovine respiratory tract response to disease.