Expression of matrix metalloproteinase-9 and -12 in porcine lung infections.

Matrix metalloproteinases (MMPs) play a variety of roles during organogenesis, in the immune response and during acute and chronic diseases as well as in tissue remodelling. During the last decade, the pig has become used increasingly as a model for human diseases; however, studies on the expression of porcine MMPs are limited. In the present study species-specific antibodies were produced to investigate the expression of MMP-9 and MMP-12 immunohistochemically in lungs from pigs infected with Actinobacillus pleuropneumoniae, Pasteurella multocida and Staphylococcus aureus. The immunolabelling of lung tissues (one infected and one control pig representing each infection) was evaluated for cellular distribution and intensity, which was scored semiquantitatively. When compared with healthy, non-infected controls, the expression of both MMP-9 and MMP-12 was higher in infected lungs. The highest expressions were seen in the alveolar epithelium (MMP-9) and alveolar macrophages (MMP-12). These results are in accordance with studies of human lungs.

Lymphocyte development requires S-nitrosoglutathione reductase.

NO is critical to immunity, but its role in the development of the immune system is unknown. In this study, we show that S-nitrosoglutathione reductase (GSNOR), a protein key to the control of protein S-nitrosylation, is important for the development of lymphocytes. Genetic deletion of GSNOR in mice results in significant decrease in both T and B lymphocytes in the periphery. In thymus, GSNOR deficiency causes excessive protein S-nitrosylation, increases apoptosis, and reduces the number of CD4 single-positive thymocytes. Lymphopenia and increase in S-nitrosylation and apoptosis in GSNOR-deficient mice are largely abolished by genetic deletion of inducible NO synthase. Furthermore, the protection of lymphocyte development by GSNOR is apparently intrinsic to hematopoietic cells. Thus, GSNOR, likely through regulation of S-nitrosylation and apoptosis, physiologically plays a protective role in the development of the immune system.

Pattern of cerebrospinal fluid lactic dehydrogenase during treatment of bacterial meningitis.

Bacterial and aseptic meningitis are characterized by distinctive lactic dehydrogenase isoenzyme patterns. No studies have quantified the dynamics of lactic dehydrogenase isoenzyme distribution during treated bacterial meningitis. We used a retrospective case-series design, and reviewed files of all neonates with bacterial meningitis who attended our pediatric tertiary medical center for 8 years period. We identified neonates in whom a repeated lumbar puncture was indicated. Findings of cerebrospinal fluid analysis, including levels of lactic dehydrogenase isoenzymes, were compared with an age-matched reference group. In two patients with meningitis, lumbar puncture with cerebrospinal fluid analysis was repeated because of inadequate response to treatment or initially obscure etiologic pathogens. Both patients had initially low levels of lactic dehydrogenase-1 and lactic dehydrogenase-2 and high levels of lactic dehydrogenase-4 and lactic dehydrogenase-5, similar to other patients with bacterial meningitis. The distribution pattern of lactic dehydrogenase isoenzyme normalized after adequate antibiotic treatment. In light of the encouraging results in these two patients, further studies are warranted regarding the value of lactic dehydrogenase isoenzyme measurements for follow-up purposes and for evaluations of response to treatment.

In vivo production of neuraminidase by Pasteurella haemolytica in market stressed cattle after natural infection.

Pasteurella haemolytica (Ph) is the most important cause of the bovine acute fibrinohemorrhagic pneumonia that occurs in market stressed calves after shipment to feedyards. Recent characterization of neuraminidase production by these organisms has shown that all 16 serotypes produce an immunologically similar form of the enzyme. Anti-neuraminidase antibody against PhA1 and PhA6 was determined in 101 2- to 5-month-old calves, on their farms of origin, at the order buyer barn (OBB), and through 28 days in the feedyard. Half of the calves were vaccinated with a killed Ph serotype-A1 (PhA1) product. Nasal secretion and tonsil wash specimens were cultured for Ph and Pasteurella multocida (Pm). Serum antibody against PhA1 and PhA6 was measured by indirect hemagglutination (IHA), and anti-neuraminidase antibody was determined by the neutralization assay. At the feedyard, 73 calves had respiratory tract disease. IHA values ranged between 1:2 and 1:1024 for PhA1 and between 1:2 and 1:512 for Ph serotype A6 (PhA6). Forty-two, 24, and 28% of the calves were infected with PhA1, PhA6, and Pm, respectively. Ninety-six percent of the calves experienced an increase in anti-PhA1 neuraminidase antibody when sera drawn on feedyard day 28 were compared with sera drawn on the farm. These data demonstrate that the enzyme neuraminidase is produced in vivo in market stressed cattle after a natural Ph infection.

In vivo production of neuraminidase by Pasteurella multocida A:3 in goats after transthoracic challenge.

Six goats were injected transthoracically with live Pasteurella multocida A:3 to examine if an extracellular enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 6) each received 1 ml of live 7.5 x 10(4) cfu of P. multocida mixed with polyacrylate beads transthoracically in the left lung on day 0 and 1 ml of live P. multocida (2.2 x 10(8) cfu) mixed with polyacrylate beads transthoracically in the left lung on day 22. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 22. Serum was obtained from all animals on days 0, 7, 14, 22, 29, and 36. Preimmune sera from all animals showed no detectable antibody to P. multocida A:3 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals demonstrated a significant antibody titer against the P. multocida A:3 neuraminidase. On day 36, serum samples from the six infected animals possessed complete enzyme-neutralizing activity. Anti-neuraminidase antibody could be detected as early as day 14 in the infected animals. These data show that neuraminidase is produced in vivo during an active P. multocida A:3 lobar infection.

Serial pleural fluid analysis in a new experimental model of empyema.

Prior attempts to create an animal model of empyema by direct inoculation of bacteria alone into the pleural space have been unsuccessful. The animals either died of overwhelming sepsis or cleared the infection from the pleural space without development of an empyema. We hypothesized that injection of bacteria with a nutrient agar into the pleural space would allow the bacteria to remain in the pleural space for an extended time period, permitting an empyema to develop. The bacterium Pasteurella multocida in brain heart infusion (BHI) agar was injected into the right hemithorax of 12 New Zealand white male rabbits. Our preliminary studies showed that the animals died in less than 7 days if they were not given parenteral antibiotics. For this reason, the rabbits were given penicillin, 200,000 U, IM, every 24 h starting 24 h after bacterial injection. Pleural fluid was sampled by thoracentesis at 12, 24, 48, 72, and 96 h after bacterial injection. Pleural fluid pH, glucose, lactate dehydrogenase (LDH), leukocyte count, and Gram's stain and culture (in one half of the animals) were obtained at each time point. Pleural biopsy specimens were obtained at autopsy after 96 h. The mean pleural fluid pH reached a nadir of 7.01 at 24 h and remained less than 7.1 throughout the experiment. The mean pleural fluid glucose level reached a nadir of 10 mg/dL at 24 h. The mean pleural fluid LDH peaked at 21,000 IU/L at 24 h and the mean pleural fluid leukocyte count peaked at 12 h with a value of 67,000 cells per cubic millimeter. Gram's stains revealed organisms and cultures were positive for growth in all animals at 12 and 24 h. Some animals had positive Gram's stains and growth on cultures up to 72 h after bacterial injection. At autopsy, all rabbits injected with bacteria had gross pus in the right pleural space and had developed a thick pleural peel. Microscopic specimens of the pleura revealed large numbers of leukocytes (primarily polymorphonuclear lymphocytes) with invasion of the adjacent lung and chest wall. In conclusion, this model more closely mimics the empyema that occurs in humans, relative to previous animal models. This model appears appropriate for additional randomized studies in which different methods for the treatment of empyema can be evaluated.

In vivo production of neuraminidase by Pasteurella haemolytica A1 in goats after transthoracic challenge.

Nine goats were injected transthoracically with Pasteurella haemolytica A1 to determine if an extracellular bacterial enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 9) each received 1 ml of 7.25 x 10(5) live P. haemolytica A1 cells in polyacrylate beads transthoracically in the left lung on days 0 and 21. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 21. Serum was obtained from all animals on days -4, 3, 7, 14, 21, 24, and 32. Preimmune serum from all animals showed no detectable antibody to P. haemolytica A1 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals possessed a significant antibody concentration in response to the P. haemolytica A1 neuraminidase. On day 32, serum samples from the nine infected animals possessed enzyme neutralizing activity that ranged from 62% to 100%. Anti-neuraminidase antibody could be detected as early as day 14 by the enzyme neutralization assay. These data demonstrate that the enzyme neuraminidase is produced in vivo during an active P. haemolytica A1 lobar infection.

Increased elastase activity in nasal mucus associated with nasal colonization by Pasteurella haemolytica in infectious bovine rhinotracheitis virus-infected calves.

Four healthy calves were inoculated with Pasteurella haemolytica serotype 1 by instillation of a broth culture into the middle nasal meatus of the left nostril. Four weeks later, calves were exposed to infectious bovine rhinotracheitis virus by aerosol into both nostrils. All calves became ill, from approximately day 3 through day 10 after virus exposure, and shed increased amounts of nasal mucus. Two calves were induced to shed P haemolytica by the virus infection, and 2 calves required reinoculation with P haemolytica for nasal passages to become actively colonized. Elastase activity in nasal mucus increased about 15-fold within 3 days and peaked about 60-fold over baseline by 7 days after virus exposure. Activity of N-acetyl-beta-D-glucosaminidase, a measure of cell damage and serum leakage, increased slightly by day 3 and reached plateau on day 5, almost threefold over baseline activity. Protein and carbohydrate content increased at a rate similar to that of N-acetyl-beta-D-glucosaminidase activity with about 12-fold and sixfold increases, respectively. None of the variables returned to baseline by 19 days after virus exposure. Increased elastase activity preceded colonization by P haemolytica and decreasing elastase activity preceded decreasing P haemolytica concentration in the nasal secretions. A causal relation between elastase activity and P haemolytica colonization could be mediated by cleavage of epithelial cell surface fibronectin and exposure of receptors.

[Glutathione and superoxide dismutase redox system in the development of toxic-infectious shock caused by Yersinia pestis toxin]. / Fermentnaia redoks-sistema glutationa i superoksiddistimustazy v dinamike chumnogo toksiko-infektsionnogo shoka.

The changes in the glutathione-dependent and superoxide dismutase (SOD) enzymatic activity in the rat lungs and liver tissues have been studied after the administration of plague murine toxin (LD100). It has been found out the early toxic effect in 1h in the lungs: 35% SOD and glutathione peroxidase (tributyl hydroperoxide) (GP) decrease, 87% glutathione reductase (GR) increase along with two-hold ascent of ratio GR/Glutathione-S-transferase (GT), GR/GPs. The fundamental ratio GR/GT.GPs rises in 1h 3.7 times and then falls below standard rate (5h). This is the evidence of the lungs antioxidant system potential power exhaustion. It has been established that in the liver, 4 times SOD activity increases in 2h after the toxin injection, and 1.5 times GP (tributyL) hydroperoxide) activity ascends in 1h. The ratio increase (150% for SOD/GP-H2O2 in 2h, 114% for GR/GP (tributyl hydroperoxide) and 61% for GR/GT in 5h) indicates the stable unbalance of this system. The pathogenetic significance of detoxication system disturbances in the lungs and liver tissues under the murine toxin influence is discussed.

Selenium effects on glutathione peroxidase and the immune response of stressed calves challenged with Pasteurella hemolytica.

The present study was conducted to determine whether a marginal Se deficiency affects health, blood characteristics and the immune response of calves subjected to stresses associated with weaning, shipping (332 km) and Pasteurella hemolytica inoculation. Treatments were 1) -Se, 2) -Se/P. hemolytica, 3) +Se (.1 mg Se/kg feed) and 4) +Se/P. hemolytica. Previous Se intake was controlled; dams of -Se calves were fed diets marginally deficient in Se (.03 to .05 mg/kg), whereas dams of +Se calves received a s.c. injection of 30 mg Se (as sodium selenite) every 60 d. Calves were inoculated with P. hemolytica intratracheally on d 3 following weaning and transport. Inoculation with P. hemolytica increased (P less than .05) body temperatures, platelet counts, serum IgM concentrations and serum antibody titers and decreased serum albumin concentrations at 4 to 7 d postinoculation. Weight gains for the 21-d study were not affected by Se status, although whole blood and plasma glutathione peroxidase (GSH-Px) were higher (P less than .05) for +Se calves. Plasma GSH-Px increased (P less than .01) in calves showing signs of morbidity. Increases in plasma GSH-Px were correlated positively with body temperature. Serum IgM concentrations were higher (P less than .05) in +Se calves on d 17, but Se-supplemented calves had lower (P less than .05) anti-P. hemolytica titers on d 17 than -Se calves. Selenium status did not affect body temperatures, plasma creatine phosphokinase or serum IgG and albumin concentrations. These results indicate that Se status can affect IgM concentrations following stress.

Lactate dehydrogenase isoenzymes in the lungs of sheep with acute and chronic pneumonia.

Total lactate dehydrogenase and the absolute and percentage levels of its isoenzymes were measured in lung lesions and macroscopically normal areas of lung from lambs with chronic proliferative exudative pneumonia and acute pasteurella pneumonia. Lung lesions had a higher total enzyme activity which was associated mainly with increases in the activity of the LDH4 and LDH5 isoenzymes, particularly in chronic pneumonia, and gave lung lesions a considerable potential for altering the serum isoenzyme distribution. Thus, the nature of any changes in the serum isoenzyme distribution will depend on whether the isoenzymes are released from abnormal or normal areas of lung. This appears to be the first report on lactate dehydrogenase isoenzymes in ovine pneumonia.

Lung lysophospholipase activity in specific-pathogen-free rats infected with Pasteurella pneumotropica or Mycoplasma pulmonis.

The effects of Pasteurella pneumotropica and Mycoplasma pulmonis infections in specific-pathogen-free rats were studied to determine whether or not bacterial infections could cause an increase in rat lung lysophospholipase activity and/or changes in bone marrow eosinophil levels. Lung lysophospholipase activity levels of M. pulmonis-infected rats were elevated with increasing infection dosages, but enzyme levels were not accompanied by a lung tissue eosinophilia or an increase in bone marrow eosinophils. Rats infected with P. pneumotropica showed neither an increased lung lysophospholipase activity level nor an increased tissue or bone marrow eosinophilia.

Influence of bacterial infection on serum enzymes of white rats.

Infection of white rats with Francisella tularensis (Pasteurella tularensis) and Salmonella typhimurium and exposure to the endotoxin of S. typhimurium stimulated significant increases in various serum enzymes including aldolase, lactate dehydrogenase, phosphohexose isomerase, isocitrate dehydrogenase, and glutamate-oxalacetate transaminase. The rates of changes in enzymatic activity after infection were directly related to the size of infecting dose and to the type of infective agent employed. Tularemic infection stimulated excessive changes in enzyme activity, whereas salmonellosis and endointoxication elicited less pronounced alterations of relatively short duration. Changes observed in serum enzymes after exposure to these agents reflect the severe liver damage and extensive systemic involvement noted in tularemia as opposed to more localized and less intensive tissue damage occurring during salmonellosis and endointoxication.