An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs.

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.

Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer.

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1, 608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein.

The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay. The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenced. Alignment of the sequences revealed conserved terminal and variable middle regions, which divided the reference strains into four distinct groups. Primers were selected from the conserved 5' and 3' termini of the gene. A 950-bp amplicon was obtained from each of 102 tested field isolates of A. pleuropneumoniae obtained from lungs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of the A. pleuropneumoniae isolates, which all showed the expected DNA sequence characteristic of the serotype. To test the specificity of the reaction, 23 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.

Chemically reactive intermediates and pulmonary xenobiotic toxicity.

After reading this lengthy review, the reader may consider it foolhardy to attempt to summarize the data contained herein. If so, the author and the reader are in complete accord. I set out to integrate, wherever possible, pathological changes in tissues and cells as determined by light, scanning, and TEM with chemical, biochemical, molecular, biological, and genetic techniques. I am not so naive as to equate correlation with causation, but in experimental biology, one is often compelled to rely on a number correlations to suggest causation. These data may also act as a stepping-off point helping the investigator design experiments to support/confirm or refute/disprove the hypothesis under investigation. Science is rarely black and white but rather gray, and the young investigator must be wary of scientists who argue their points too vociferously, too loudly, or too selectively. The truth in science is often a Gordian knot.

Comparative activity of N-(4-hydroxyphenyl)-all-trans-retinamide and alpha-difluoromethylornithine as inhibitors of lymphoma induction in PIM transgenic mice.

The activities of the retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR) and the polyamine synthesis inhibitor, alpha-difluoromethylornithine (DFMO), as inhibitors of lymphoma induction in PIM transgenic mice were evaluated. Lymphoma was induced in male PIM mice by a single intraperitoneal injection of 50 mg N-ethyl-N-nitrosourea (ENU) per kg body weight. Continuous dietary administration of 4-HPR (391, 196 or 98 mg/kg diet) or DFMO (1000, 500 or 250 mg/kg diet) was initiated immediately after ENU administration, and was continued until the end of the study at 35 weeks. At 20 weeks post-ENU, the high dose of 4-HPR reduced both lymphoma incidence and associated mortality. However, the protection conferred by 4-HPR represented a delay rather than an inhibition of neoplastic development, since both lymphoma incidence and mortality at study termination were similar in dietary controls and all groups treated with 4-HPR. DFMO had no effect on lymphoma incidence, latency or mortality at any point in the study. These results suggest that 4-HPR or other retinoids may be effective in the prevention of lymphoma induction, whereas inhibition of polyamine biosynthesis does not appear to present a useful mechanistic target for the chemoprevention of lymphoid neoplasia. The PIM transgenic mouse provides a useful in vivo model for the rapid evaluation of chemopreventive agents.

Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils.

A PCR for the detection of Actinobacillus pleuropneumoniae was evaluated. All of 102 field, isolates of A, pleuropneumoniae reacted in the PCR by amplification of a 985 bp product. No PCR amplification product was observed when examining strains of A. ureae, A. capsulatus, A. hominis, A. equuli, A, rossii, A. suis, Escherichia coli, Bordetella bronchiseptica. Streptococcus suis, Pasteurella haemolytica, Pasteurella multocida, Haemophilus parasuis, Haemophilus taxon Minor group, Haemophilus taxon D/E and haemophilus taxon F. Amplification of a 985 bp product was, however, observed when testing strains of A. lignieresii. The lower detection limit of the PCR test was 10(3) A. pleuropneumoniae CFU/PCR test tube and was not affected by addition of 10(6) E. coli CFU/PCR test tube. Mixed bacterial cultures from tonsils of 101 pigs from 9 different herds were tested by culture and by PCR using four different bacteriological media. While 65% reacted positive in the PCR only 23% were positive by culture, thereby suggesting a superior sensitivity of the PCR test to that of culture. The use of selective media, large inoculum and incubation for 48 h gave the highest number of positive PCR reactions from mixed bacterial cultures. Tonsil cultures from 50 pigs from an A. pleuropneumoniae-negative herd did not react in the PCR. The results show that PCR on mixed bacterial cultures from tonsils may be a highly sensitive method for the detection of A. pleuropneumoniae in pig herds.

Exceptional chemopreventive activity of low-dose dehydroepiandrosterone in the rat mammary gland.

To determine if the chemopreventive activity of dehydroepiandrosterone (DHEA) in the rat mammary gland can be dissociated from its toxicity, two studies were conducted in which low doses of DHEA were administered alone and in combination with other agents to rats treated with N-methyl-N-nitrosourea. Beginning 1 week prior to administration of 35 mg N-methyl-N-nitrosourea per kg body weight, groups of 20 female Sprague-Dawley rates were fed AIN-76A diet supplemented with DHEA alone (800 or 400 mg/kg diet), DHEA + tamoxifen (80 or 40 microgram/kg diet), DHEA + carbenoxolone (3500 or 1750 mg/kg diet), or DHEA + tamoxifen + carbenoxolone. When administered alone at either 800 or 400 mg/kg diet, DHEA reduced mammary cancer incidence from >70% in dietary controls to 0%; mammary cancer incidence from >70% in dietary controls to 0%; mammary cancer incidence in all DHEA combination regimens was also < or = 5%. The dose levels of DHEA used induced no toxicity or alteration in body weight gain. These results indicate that dietary supplementation with low doses of DHEA has chemopreventive efficacy greater than or equal to that of endocrine ablation. This protection may be mediated by the induction of differentiation in the mammary parenchyma.

A possible role for membrane lipid peroxidation in anthracycline nephrotoxicity.

Adriamycin causes both glomerular and tubular lesions in kidney, which can be severe enough to progress to irreversible renal failure. This drug-caused nephrotoxicity may result from the metabolic reductive activation of Adriamycin to a semiquinone free radical intermediate by oxidoreductive enzymes such as NADPH-cytochrome P-450 reductase and NADH-dehydrogenase. The drug semiquinone, in turn, autoxidizes and efficiently generates highly reactive and toxic oxyradicals. We report here that the reductive activation of Adriamycin markedly enhanced both NADPH- and NADH-dependent kidney microsomal membrane lipid peroxidation, measured as malonaldehyde by the thiobarbituric acid method. Adriamycin-enhanced kidney microsomal lipid peroxidation was diminished by the inclusion of the oxyradical scavengers, superoxide dismutase and 1,3-dimethylurea, and by the chelating agents, EDTA and diethylenetriamine-pentaacetic acid (DETPAC), implicating an obligatory role for reactive oxygen species and metal ions in the peroxidation mechanism. Furthermore, the inclusion of exogenous ferric and ferrous iron salts more than doubled Adriamycin-stimulated peroxidation. Lipid peroxidation was prevented by the sulfhydryl-reacting agent, p-chloromercuribenzenesulfonic acid, by omitting NAD(P)H, or by heat-inactivating the kidney microsomes, indicating the requirement for active pyridine-nucleotide linked enzymes. Several analogs of Adriamycin as well as mitomycin C, drugs which are capable of oxidation-reduction cycling, greatly increased NADPH-dependent kidney microsomal peroxidation. Carminomycin and 4-demethoxydaunorubicin were noteworthy in this respect because they were three to four times as potent as Adriamycin. In isolated kidney mitochondria, Adriamycin promoted a 12-fold increase in NADH-supported (NADH-dehydrogenase-dependent) peroxidation. These observations clearly indicate that anthracyclines enhance oxyradical-mediated membrane lipid peroxidation in vitro, and suggest that peroxidation-caused damage to kidney endoplasmic reticulum and mitochondrial membranes in vivo could contribute to the development of anthracycline-caused nephrotoxicity.

In vitro studies on the metabolism and covalent binding of [14C]1,1-dichloroethylene by mouse liver, kidney and lung.

The metabolism and covalent binding of 1,1-dichloro[1,2-14C]ethylene (DCE) to subcellular fractions of liver, kidney and lung of C57BL/6N mice have been investigated in vitro. Covalent binding was NADPH- and cytochrome P-450-dependent. The microsomal fraction bound more radiolabel than any other subcellular fraction, and the levels of covalent binding in cell fractions correlated well with their cytochrome P-450 content. Covalent binding by mouse liver and lung microsomes also reflected their cytochrome P-450 content. However, although mouse kidney microsomes contained twice as much total cytochrome P-450 as the lung, no detectable covalent binding of DCE-derived radioactivity occurred in kidney. Omission of NADPH, heat inactivation of microsomes, carbon monoxide, addition of SKF-525A, piperonyl butoxide or reduced glutathione (GSH), all inhibited (40-90%) covalent binding of radiolabel to liver and lung microsomes. The absence of O2 (incubation under N2) did not greatly affect the metabolism and covalent binding. Pretreatment of mice with various inducers, phenobarbital (PB), beta-naphthoflavone (beta-NF), pregnenolone 16 alpha-carbonitrile (PCN) and 3-methylcholanthrene (3-MC), evoked increases in total liver microsomal cytochrome P-450 content (2-fold) and corresponding increases in covalent binding (3-fold). However, microsomes from PCN-treated mice showed only a 50% increase in DCE binding. Kidney microsomes from control, PB-, and beta-NF-pretreated mice were incapable of covalent binding of radiolabel but those from PCN- and 3-MC-pretreated mice showed levels of binding similar to untreated mouse lung microsomes. It is proposed that the nephrotoxicity of DCE may be due to translocation of reactive metabolites from the liver to the kidney.

Protection by methylprednisolone against butylated hydroxytoluene-induced pulmonary damage and impairment of microsomal monooxygenase activities in the mouse: lack of effect on fibrosis.

The effects of the synthetic corticosteroid methylprednisolone (MP; 30 mg/kg, s.c. given twice daily for 3 days), on the pneumotoxic effects of a single dose of butylated hydroxytoluene (BHT; 400 mg/kg, i.p.) over a 10 day experimental period was investigated in male C57BL/6N mice. BHT alone caused time-dependent alveolar hypercellularity, inflammatory infiltration, alveolar septal thickening and hypercellularity of the bronchiolar epithelium, reaching a maximum by day 5 with some degree of recovery by day 10. The pulmonary monooxygenase activities reflected the degree of alveolar damage and Clara cell abnormality with time; reductions in monooxygenase activities occurred and minimum levels (7-15% of control) were reached by day 5 and again a trend towards recovery by day 10. MP administered 0, 24 and 48 hr after BHT treatment partially protected mice from these effects of BHT in a distinctly time-dependent fashion; the degree of protection decreased as the time between BHT challenge and MP treatment increased. Although MP alone did not morphologically affect Clara and alveolar cells, it increased, decreased or had no effect on the monooxygenase activities. About 25% of the mice that received BHT alone died by day 5 and 50% by day 10. MP completely blocked the lethal effects of BHT by day 5 and reduced the deaths to between 15% and 25% by day 10. Interestingly, MP did not protect against the BHT-induced pulmonary fibrosis, measured as total lung hydroxyproline content, irrespective of the time between BHT challenge and MP treatment. MP alone did not cause any deaths nor increase lung hydroxyproline content.

Studies on the distribution and covalent binding of 1,1-dichloroethylene in the mouse. Effect of various pretreatments on covalent binding in vivo.

The distribution and covalent binding of a single dose of [1,2-14C] 1,1-dichloroethylene (DCE; 125 mg/kg, i.p.) was studied in male C57Bl/6N mice. Total radioactivity was distributed in whole homogenates of all tissues studied, with peak levels occurring within 6 hr. Covalent binding of radioactive material peaked at 6-12 hr in all tissues, and highest levels were found in kidney, liver, and lung with smaller amounts in skeletal muscle, heart, spleen, and gut. Covalent binding in kidney, liver, and lung fell to 50% of peak levels in about 4 days. Between 12 hr and 4 days after DCE administration, 70-100% of total radioactivity present in homogenates of kidney, liver, and lung was covalently bound. The three tissues showed a similar spread in total radioactivity in subcellular fractions 24 hr after exposure to DCE; most of the radioactivity was covalently bound (60-100%) and distributed fairly uniformly with a slight tendency to concentrate in the mitochondrial fraction. Phenobarbital (PB) and 3-methylcholanthrene (3-MC) pretreatments increased the covalent binding in the liver and lung but had no effect in the kidney. Piperonyl butoxide and SKF-525A decreased the covalent binding in liver and lung, but the latter increased binding in the kidney while the former decreased it. Diethylmaleate administration increased the covalent binding (2- to 3-fold) in all three tissues as well as increasing lethal toxicity. These results are consistent with the view that DCE is metabolized to some reactive intermediate(s) which may be detoxified by conjugation with glutathione.

The utility of changes in cardiac weight as an index of drug-induced cardiotoxicity.

The effects of toxic doses of various drugs and of food or water deprivation upon heart weights of mice were evaluated over a four day period to test the validity of the hypothesis that changes in cardiac weights are indicators of cardiotoxicity. Drugs included in the study were actinomycin-D, methotrexate, 5-fluorouracil, adriamycin, daunomycin, N-dimethyladriamycin, N-trifluoroacetyladriamycin-14-valerate, isoproterenol, atropine, and acetylsalicylic acid. Additional groups of mice served as vehicle controls, or were deprived of food or water for the duration of the experiment to control for the anorexia and dehydration accompanying treatment with antineoplastic drugs. Body weights were taken at the start of the experiment (day 0), day 2, and day 4 (just prior to sacrifice). Heart ventricle wet weights were determined immediately, and dry weights after thorough desiccation of the samples. Statistical evaluation of the weights revealed that there were no ventricular weight changes unique to any particular drug, and that decreases in heart weights correlated well with decreases in body weights, thereby reflecting the general toxicities of the drugs, including inanition, and not any specific cardiotoxicities.

Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin.

Mitochondrial degeneration is a consistently prominent morphological alteration associated with adriamycin toxicity which may be the consequence of adriamycin-enhanced peroxidative damage to unsaturated mitochondrial membrane lipids. Using isolated rat liver mitochondria as an in vitro model system to study the effects of the anticancer drug adriamycin on lipid peroxidation, we found that NADH-dependent mitochondrial peroxidation--measured by the 2-thiobarbituric acid method--was stimulated by adriamycin as much as 4-fold. Marker enzyme analysis indicated that the mitochondria were substantially free of contaminating microsomes (less than 5%). Lipid peroxidation in mitochondria incubated in KCl-Tris-HCl buffer (pH 7.4) under an oxygen atmosphere was optimal at 1-2 mg of mitochondrial protein/ml and with NADH at 2.5 mM. Malonaldehyde production was linear with time to beyond 60 min, and the maximum enhancement of peroxidation was observed with adriamycin at 50-100 microM. Interestingly, in contrast to its stimulatory effect on NADH-supported mitochondrial peroxidation, adriamycin markedly diminished ascorbate-promoted lipid peroxidation in mitochondria. Superoxide dismutase, catalase, 1,3-dimethylurea, reduced glutathione, alpha-tocopherol and EDTA added to incubation mixtures inhibited endogenous and adriamycin-augmented NADH-dependent peroxidation of mitochondrial lipids, indicating that multiple species of reactive oxygen (superoxide anion radical, hydrogen peroxide and hydroxyl radical) and possibly trace amounts of endogenous ferric iron participated in the peroxidation reactions. In submitochondrial particles freed of endogenous defenses against oxyradicals, lipid peroxidation was increased 7-fold by adriamycin. These observations suggest that some of the effects of adriamycin on mitochondrial morphology and biochemical function may be mediated by adriamycin-enhanced reactive oxygen-dependent mitochondrial lipid peroxidation.

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice.

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.

Selective damage to nonciliated bronchiolar epithelial cells in relation to impairment of pulmonary monooxygenase activities by 1,1-dichloroethylene in mice.

A single ip dose of 1,1-dichloroethylene (DCE) to mice (125 mg/kg) caused a reduction within 24 hr in cytochrome P-450 and related monooxygenases in lung microsomes, with no corresponding changes in liver and kidney microsomes. Light microscopy revealed that at 24 hr, DCE caused a highly selective and complete loss of the bronchiolar nonciliated (Clara) cells at all levels of the tracheobronchial tree. Electron microscopy showed that at this time, the bronchiolar luminal surface was covered by flattened, elongated ciliated cells. Within 24 hr total microsomal cytochrome P-450 and NADPH cytochrome c reductase were maximally reduced to about 50% of control and cytochrome P-450-dependent enzyme activities decreased to about 60% of control. By contrast, coumarin 7-hydroxylase was reduced to approximately 10% of control within 4 days. Since pulmonary coumarin 7-hydroxylase has been shown to reside almost exclusively in the Clara cells, this finding is in agreement with the observed extensive necrosis of the Clara cells. The return of lung microsomal P-450-linked enzyme activities took between 3 and 6 weeks and was paralleled by a corresponding slow reappearance of the bronchiolar Clara cells.