Expression profiling reveals differences in immuno-inflammatory gene expression between the two disease forms of sheep paratuberculosis.

Paratuberculosis is a chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP); infection of sheep results in two disease forms - paucibacillary (tuberculoid) and multibacillary (lepromatous) associated with the differential polarization of the immune response. In addition the majority of MAP-infected animals show no pathology and remain asymptomatic. Microarray and real-time RT-qPCR analyses were used to compare gene expression in ileum from sheep with the two disease forms and asymptomatic sheep, to further understand the molecular basis of the pathologies. Microarrays identified 36 genes with fold-change of >1.5 and P< or = 0.05 in at least one comparison; eight candidates were chosen for RT-qPCR validation. Sequence analysis of two candidates, CXCR4 and IGFBP6, identified three SNPs in each; five were found in all three forms of disease and showed no significant relationship to pathological type. The IGFBP6 G(3743) A SNP was not detected in asymptomatic sheep. The data show that the two forms of disease are associated with distinct molecular profiles highlighted by the differential expression of chemokine and chemokine receptor transcripts, the protein products of which might be implicated in the different cell infiltrates of the pathologies. The cells within the lesions also show evidence of abnormal activation; they express high levels of cytokine transcripts but have reduced expression levels of transcripts for T cell receptor associated molecules.

Gene expression profiling of ovine keratinocytes stimulated with Psoroptes ovis mite antigen--a preliminary study.

Sheep scab is caused by the noninvasive mite, Psoroptes ovis, which initiates a profound pro-inflammatory skin response leading to lesion development. To investigate these early events between the skin and the parasite, primary ovine epidermal keratinocyte cultures were generated and challenged with mite derived antigens. The kinetics of the mRNA response of these cells were monitored by microarray. The results indicated that the cells responded within 1 h of challenge, with a significant increase in the pro-inflammatory cytokine IL-8. This result was confirmed by real-time RT-PCR, and showed that IL-8 up-regulation was maximal at 1 h but declined to pre-stimulation levels at 24 and 48 h. The IL-8 mRNA response to mite wash antigens containing secretory and/or excretory proteins was also investigated and compared to the response to whole mite antigen. These studies revealed that the mite wash antigen, at a challenge dose of 10 microg/mL, was markedly more potent and induced significantly higher levels of IL-8 mRNA than the same concentration of whole mite antigen. These results are discussed in relation to mite establishment and survival on the ovine host.

Contribution of respiratory burst activity to innate immune function and the effects of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro.

The mechanisms of interaction between phagocytes and different bacteria that help resolve lung infections or contribute to lung pathology are poorly defined. Alveolar phagocytes (resident macrophages and recruited neutrophils) make a major contribution to innate immunity by mounting a respiratory burst that helps kill internalised bacteria. However, this ability may be altered during or after exposure to infection. This review considers the application and limitations of a variety of analytical methods for oxygen-dependent mechanisms of respiratory burst in phagocytes initiated by soluble and particulate activators. Particular reference is given to the study in vitro of phagocytes from healthy and diseased ruminants during either natural infection with Mycobacterium avium paratuberculosis or experimental infection with Pasteurella multocida or Mannheimia haemolytica.

Salivary concentration of secretory leukocyte protease inhibitor, an antimicrobial protein, is decreased with advanced age.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) exhibits antimicrobial activities that, in addition to other well-characterized proteins such as lysozyme and lactoferrin, is thought to play a critical role in mucosal defenses. Although elderly individuals are particularly susceptible to mucosal infections, salivary production of SLPI has not been assessed in an aged cohort. OBJECTIVES: Hypothesizing that oral SLPI concentrations are reduced with advanced age, this cross-sectional study compared SLPI concentrations to concentrations of lysozyme, lactoferrin and total protein in unstimulated salivary secretions of healthy, community-dwelling 79+-year-old and younger adults. METHODS: Study participants were 45 non-hospitalized dentate adults aged 79-89 (23 elderly) or 21-51 years (22 non-elderly). Home-based interviews and clinical examinations determined dentate status and confirmed the absence of dentures, oral mucosal disease, anti-infective medication use, irradiation therapy for head and neck cancer and self-perceived xerostomia. Whole unstimulated saliva was collected from all subjects and analyzed for antimicrobial protein concentration by enzyme-linked immunosorbent assay and for total protein content by the bicinchoninic acid method. Bivariate and multivariate (generalized linear modeling) analyses evaluated the relationships between age, gender and salivary protein concentrations. RESULTS: Mean salivary levels of SLPI and lysozyme were lower in elderly compared with non-elderly subjects (p < 0.001), unlike lactoferrin and total protein levels. Similar results were obtained when concentrations of the individual proteins were normalized to the total protein concentration, suggesting that glandular production of SLPI and lysozyme preferentially decreases with aging. Gender differences were detected only for SLPI concentrations; males had lower SLPI levels than females regardless of age (p < 0.01). Generalized linear models confirmed that age (p < 0.001) and gender (p < 0.05) were each associated with the SLPI concentration and together accounted for 50% of the variation in SLPI concentration in this population. CONCLUSION: These findings indicate that SLPI production is diminished among healthy community-dwelling older adults, particularly elderly males. Further investigation should determine the impact of decreased local SLPI production on the increased risk of oral mucosal disease with advanced age.

Comparison of three salivary flow rate assessment methods in an elderly population.

Measuring salivary flow rates among the frail elderly is a challenge. The currently used spit collection method requires levels of time and cooperation that often may exclude the frail elderly who are at high risk for salivary compromise. A measurement method that is not only valid and reliable, but also feasible and acceptable is needed for use in population studies of compromised adults. This study compared two salivary flow rate assessment methods using a suction machine against the currently accepted spit collection method in an elderly population aged 75 and older. Three methods of flow rate (g/min) assessment were compared at three time periods among 16 elders (mean age 86.6 years). Flow rates using the 2-min open suction method compared well with the 10-min spit method (r=0.778) but the 2-min closed suction method did not (r=0.158). Reliability evaluation of the open suction method and the spit method was assessed using a test/retest with a 1-week interval. Both methods demonstrated good comparable reliability (spit method r=0.566, P=0.01); open suction method, (r= 0.861, P<0.01). Based on a short survey questionnaire about the three methods, 11 of the 16 elderly subjects preferred the use of the suction machine to the spit method. These results indicate that the 2-min open suction method technique is a valid and reliable means of measuring salivary flow. The lower level of patient cooperation needed, the shorter time period required, and this preliminary report of its acceptability support the use of this method in future population studies of frail elders.

Metabolic effects of isoflurane on rat lungs perfused in situ.

1. The current experiments studied the effects of the inhalation anesthetic, isoflurane, on 5-hydroxytryptamine (5-HT) metabolism, protein synthesis, and angiotensin-converting enzyme activity in perfused rat lungs. 2. Under first order reaction conditions, isoflurane decreased the accumulation of tissue 5-hydroxyindoleacetic acid, the principle metabolite of 5-HT in a concentration-related, competitive, and reversible manner, indicating inhibition of endothelial 5-HT uptake. 3. In apparent contrast, isoflurane appeared to stimulate uptake of 5-HT by an imipramine-sensitive process, into a cell type unable to metabolize the parent amine. 4. Isoflurane increased absolute angiotensin-converting enzyme activity only at an inspired concentration of 5%. The anesthetic did not affect lung protein synthesis.

Effects of ketamine and fentanyl on lung metabolism in perfused rat lungs.

The effects of ketamine and fentanyl on serotonin (5-hydroxytryptamine; 5-HT) metabolism, angiotensin-converting enzyme (ACE), and protein synthesis (PS) were investigated in an isolated lung model. Rat lungs were perfused in situ with a blood-free physiological salt solution. The pulmonary vasculature was exposed to ketamine (0.005-2.1 mM) or fentanyl (1.8-4.5 microM) for up to 2 h. After 1 h, accumulation of 5-[14C]hydroxyindoleacetic acid (5-HIAA) by the lung was monitored as an index of 5-HT metabolism. ACE activity was estimated from hydrolysis of [3H]benzoylphenylalanyl-alanyl-proline, a synthetic substrate for the enzyme. [3H]phenylalanine was added to the perfusate after 1 h, and its incorporation into acid-precipitable lung protein was measured over the subsequent hour. Ketamine inhibited 5-HT uptake in a concentration-related manner. The inhibition was characterized as competitive and reversible. Fentanyl had no effect on lung 5-HIAA accumulation. Neither drug altered ACE activity or protein synthesis over the concentration ranges tested. The results indicate an action by ketamine that inhibits the 5-HT membrane-transport process. The different effects observed by ketamine and fentanyl on this process could contribute to the diverse pharmacological properties of these two drugs.

Inhibition of synaptosomal serotonin uptake by Ketalar.

The effects of the clinical preparation of ketamine, Ketalar and its preservative, benzethonium chloride on [3H]5-hydroxytryptamine uptake were studied using rat brain synaptosomes. Ketalar caused a concentration-dependent inhibition of substrate uptake by the high affinity transport site (I50 = 20.2 +/- 2.75 microM) while benzethonium chloride had no effect. Kinetic analysis indicated the inhibition to be competitive with serotonin; the apparent km (54 nM) was increased nearly two-fold at 10 microM ketamine. This action may represent a mechanism involved in ketamine anesthesia.

Anesthetic effects on 5-hydroxytryptamine uptake by rat brain synaptosomes.

As a neurotransmitter involved in modulating central nervous system nociception and awareness, 5-hydroxytryptamine (5-HT) may play an important role in the clinical sequelae of certain anesthetic compounds. Anesthetic agents are known to affect peripheral, non-neuronal 5-HT uptake but little is known about their effects on 5-HT metabolism in the central nervous system. The effects of several anesthetic compounds on 5-HT uptake were examined in synaptosomes isolated from rat brain cortex. Inhibition of this uptake process was observed by exposure to clinically relevant concentrations of the volatile anesthetics halothane, isoflurane, and enflurane. The non-volatile agent, ketamine also inhibited uptake while the narcotic fentanyl had an effect only at the highest concentrations tested. Non-volatile agents which had neither a consistent nor significant effect on synaptosomal 5-HT uptake included pentobarbital, sufentanil, and etomidate. These alterations of 5-HT metabolism could represent a mechanism that contributes to anesthetic action.

Effects of reduced pulmonary flow and hypoxia on metabolism of serotonin by rat lungs perfused in situ.

To investigate the extent to which reduced pulmonary flow may affect non-ventilatory functions of the lung, pulmonary artery pressures were altered systematically in an in vitro perfused lung preparation. Metabolic integrity of the tissue was assessed at two levels: disposition of exogenous serotonin (5-hydroxytryptamine; 5-HT) was monitored as a specific indicator of endothelial cell metabolism; and whole-tissue rates of protein synthesis and levels of ATP were evaluated as indices of general metabolic activity and energy availability. Rat lungs were perfused with recirculating cell-free buffer (37 degrees C) for 1 or 3 h at high (36) or low (3 ml.min-1.g-1) pulmonary flow; initial rates of 5-HT metabolism were measured over a subsequent 2-min interval of single-pass perfusion. Metabolism of 5-HT was inhibited and protein synthesis decreased 35% at low pulmonary flow. These changes did not appear to result directly from hypoxia, nor from the associated fall in tissue ATP. The effects of low flow were not reversed at high PO2, nor was 5-HT metabolism inhibited by restricted oxygen availability at high flow rates. After as long as 3 h exposure to a combination of low flow, ventilation (V = 0), and temperature (27 degrees C) and to the volatile anesthetic, halothane, inhibitory effects on both amine and protein metabolism were rapidly reversible. Reductions in the rate of 5-HT metabolism at reduced flow involved a decrease in the maximal velocity (Vmax: 8.0 to 2.2 nmol.min-1.g-1), without change in the apparent Km (2.6-3.2 microM) of the pathway for amine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.

Protein synthesis in perfused rat lungs: determinations based on incorporation of radioactive proline.

Functional compartmentation and metabolism of radioactive proline was evaluated to define conditions under which synthesis of lung proteins could be measured accurately based on proline incorporation. Rat lungs were perfused with Krebs-Henseleit bicarbonate buffer equilibrated with O2/N2/CO2 (20:75:5) and containing 4.5% (w/v) bovine serum albumin, 5.6 mM glucose and amino acids at plasma levels. Intracellular proline increased linearly as perfusate proline concentration was increased from 108 microM, the plasma level, to 540 or 1080 microM. At each concentration, the pool of proline which provided precursors to protein synthesis rapidly reached a steady-state specific radioactivity, but when extracellular proline was 108 microM, this pool was diluted significantly by proline from endogenous sources. At 540 or 1080 microM extracellular proline, the specific radioactivities of perfusate and intracellular proline approached equality and rates of protein synthesis calculated based on the specific radioactivity of extracellular proline compared favorably with those calculated from the specific radioactivity of phenylalanyl-tRNA. Similar results were obtained in lungs of two groups of rats in which intracellular proline concentration differed 3-fold. Thus, the contribution of endogenous proline to the pathway of protein synthesis was minimized when extracellular proline was present at high concentration. Under this condition, calculations of protein synthesis based on proline incorporation were most accurate.

Plasmalemmal metabolic activities in cultured calf pulmonary arterial endothelial cells.

We have determined kinetic characteristics of angiotensin converting enzyme, 5'-nucleotidase and transmembrane serotonin uptake and metabolism in cultured calf pulmonary arterial endothelial cells. Angiotensin converting enzyme activity was 2.8 +/- 0.03 Units/10(6) cells (N = 19; 1 Unit: amount of enzyme required to metabolize 1% of substrate, benzoyl-Phe-Ala-Pro, in 1 min under conditions of first order reaction kinetics) in confluent monolayers and 2.31 +/- 0.06 Units/10(6) cells (N = 20) in homogenates. 5'-Nucleotidase activity (substrate: 5'-AMP) was 0.25 +/- 0.01 Units/10(6) cells (N = 19) in monolayers and 0.26 +/- 0.01 Units/10(6) cells (N = 20) in homogenates. Kinetic constants for angiotensin converting enzyme were: Km = 7.6 microM, Vmax = 5.2 nmol/hour/10(6) cells and for 5'-nucleotidase: Km = 52.6 microM, Vmax = 6.3 nmol/hour/10(6) cells. These data confirm that both angiotensin converting enzyme and 5'-nucleotidase are ectoenzymes with no cytoplasmic activity. Serotonin uptake exhibited both a saturable (Km = 0.27 microM, Vmax = 17 pmol/hour/10(6) cells) and a non-saturable component.

Lung growth in response to unilateral pneumonectomy in rapidly growing rats.

The rapidity with which lung growth is initiated and completed after pneumonectomy was examined in young rats (4 wk of age; 82 g). After left pneumonectomy, the remaining lobes of the right lung grew to equal the weight of both lungs of control animals by day 7 and within 14 days increased from 366 to 968 mg. The tissue concentrations of RNA, DNA phosphate, collagen, and noncollagen proteins did not increase during the growth response. In contrast, total amounts of these constituents increased significantly in the remaining lung of pneumonectomized animals during the 1st postoperative wk and approached levels found in both lungs of sham-operated and unoperated controls by the end of the 2nd wk after pneumonectomy. Although cell size increased in control lungs during the experimental period, there was little evidence of additional cellular hypertrophy associated with compensatory lung growth. The character of the response to pneumonectomy in these rats was similar to that observed previously in older animals (320 g). Thus in spite of the higher basal rate of lung growth in the younger rats, the pattern and rapidity of compensation after pneumonectomy was similar in both age groups.

Effect of age on the accumulation of lung protein following unilateral pneumonectomy in rats.

The effects of unilateral pneumonectomy (PNX) on the net synthesis of right lung protein were investigated in vivo using three groups of rats with body weights (BW) ranging from 85 to 330 g. These data were compared to those from sham-operated and normal growing control animals. After PNX, both the 2-day lag prior to the compensatory increase in right lung mass (LW) and the subsequent rate of increase in LW and LW/BW ratio were independent of two-fold differences in the basal rate of lung growth. In all PNX groups, both right LW and LW/BW reached control values for both lungs, but in the older rats the time required for complete compensation was extended from 5 days to 12 days. The rate of net accumulation of right lung protein increased two-fold in the youngest PNX rats and 6 to 8-fold in the older animals, but when these changes were normalized to the protein content of the remaining tissue, the older rats appeared to respond to PNX less efficiently. Increased tissue levels of RNA and the resulting increased capacity of the lungs for protein synthesis could account for the accelerated rate of gain in right lung protein following PNX in both adult and young animals.

Regulation of protein synthesis in lung by amino acids and insulin.

Acute effects of amino acid availability and insulin on protein synthesis were investigated in rat lungs perfused in situ with buffer containing either 4.5% fraction V bovine serum albumin (FrV BSA), 4.5% essentially fatty acid-free (FAF) BSA, or 4.5% dextran to maintain colloid osmotic pressure. In the presence of FrV BSA, protein synthesis was unaffected by perfusion for 1 or 3 h with buffer containing no added amino acids (0X), as compared with amino acids at concentrations one (1X) or five (5X) times those in rat plasma. Regardless of the amino acid concentration, addition of insulin was without effect. Likewise, in lungs perfused for 1 h with either FAF BSA or dextran, protein synthesis was insensitive to amino acid availability or to insulin. After 3 h, however, protein synthesis decreased 34 and 37%, respectively, when these lungs were perfused in the absence of both amino acids and insulin. In both cases, the inhibition was prevented by addition of insulin to the perfusate; addition of the hormone to perfusate containing 1X amino acids or elevating perfusate amino acids to 5X did not affect protein synthesis. The deficit in protein synthesis observed in the absence of both amino acids and insulin was not accompanied by ATP depletion or by lower intracellular concentrations of amino acids. Similarly, the effect of insulin was not associated with a general elevation in intracellular amino acid concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible inhibition of protein synthesis in lung by halothane.

Alterations in the synthesis and degradation of proteins were investigated in intact lungs exposed to the volatile anaesthetic halothane. In rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6 mM-glucose, plasma concentrations of 19 amino acids and 690 microM-[U-14C]-phenylalanine and equilibrated with O2/N2/CO2 (4:15:1), protein synthesis, calculated based on the specific radioactivity of aminoacyl-tRNA, was inhibited by halothane. The anaesthetic did not affect degradation of lung proteins. The inhibition of protein synthesis was rapid in onset, dose-dependent, and quickly reversible. It did not appear to be associated with overall energy depletion, with non-specific changes in cellular permeability, or with decreased availability of amino acids as substrates for protein synthesis.

Identification of specific proteins synthesized by type II pneumocytes in primary culture.

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.

Effect of halothane on metabolism of 5-hydroxytryptamine by rat lungs perfused in situ.

The effect of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the uptake of 14C-labelled 5-hydroxytryptamine (5-HT) and its metabolism to 5-hydroxyindol-3-ylacetic acid (5-HIAA) was investigated in rat lungs perfused in situ. The rate of accumulation of 14C-labelled 5-HIAA in the tissue, monitored as an index of 5-HT metabolism, was linear with time, displayed saturation kinetics and remained stable for at least 180 min of perfusion. Exposure of the lungs to halothane (4%) for 60 min reversibly reduced production of 5-HIAA through an increase in the apparent Km for metabolism of the amine from 1.45 to 3.52 microM (P less than 0.001); the anaesthetic had no effect on the Vmax. of the process. The magnitude of the inhibition increased with time of exposure to the anaesthetic. Halothane exposure did not alter the distribution of [3H]sorbitol or [14C]5-HT, pulmonary vascular resistance, levels of ATP or the kinetics of amino acid transport in the tissue. Inhibition of protein synthesis by cycloheximide did not mimic the effect of the anaesthetic. These observations, together with those made in lungs exposed to inhibitors of 5-HT uptake and metabolism, were consistent with a halothane-mediated inhibition of 5-HT uptake, which did not appear to involve non-specific changes in membrane permeability.