Mycoplasma bovis escapes bovine neutrophil extracellular traps.

Mycoplasma bovis is a significant pathogen in bovine infections including mastitis, pneumonia, arthritis and otitis media, and is the cause of large economic losses in beef and dairy farms. During infection with M. bovis, recruited neutrophils are not sufficient to eradicate M. bovis from the infection site. The release of neutrophil extracellular traps (NETs) is one of the innate immune responses of neutrophils but the effect of M. bovis on NET formation by bovine neutrophils has not yet been clarified. The objective of our research was to examine the effect of M. bovis on NET formation and the killing activity of bovine neutrophils. We showed that NETs were not detected following stimulation of neutrophils by M. bovis alone or with Phorbol 12-myristate 13-acetat (PMA). Reactive oxygen species production is essential for NET formation but the levels in neutrophils stimulated with M. bovis at multiplicity of infections of 10, 100, and 1000 were similar to those of unstimulated cells. NET formation induced by PMA stimulated neutrophils disappeared following the addition of M. bovis but this phenomenon was not observed when ethylenediaminetetraacetic acid (EDTA) was added. M. bovis colony forming units were significantly decreased by the addition of EDTA in the presence of NETs. Our results suggested that M. bovis infection alone did not induce NETs and that M. bovis nucleases, as hypothesis-based, contributed to resistance against the killing activity of NETs.

[Mycoplasma genitalium-derived lipid-associated membrane proteins negatively regulate cytokine secretion by inducing HO-1 expression in placental trophoblast cells].

OBJECTIVE: To observe the expression of heme oxygenase-1 (HO-1) in regulation of cytokines response induced by Mycoplasma genitalium-derived lipid-associated membrane proteins (LAMPs) in placental trophoblast cells. METHODS: Placental trophoblast cells were cultured in vitro and stimulated by 0.5-5 µg/mL LAMPs for 4 to 12 hours. Expression of HO-1 mRNA and protein, and nuclear translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) were detected by real-time quantitative PCR and Western blotting, respectively. The intracellular formation of reactive oxygen species (ROS) was detected by the fluorescent probe H2DCFDA. N-acetyl-cysteine (NAC) and nuclear factor erythroid-2 related factor 2 (Nrf2) siRNA were respectively used to analyze the roles of ROS and Nrf2 in mediating HO-1 expression. Finally, placental trophoblast cells were transfected with HO-1 siRNA, or preincubated by the HO-1 agonist cobalt protoporphyrin (CoPP) or its inhibitor zinc protoporphyrin (ZnPP), and LAMPs-induced secretion of TNF-α and IL-1ß were detected by ELISA. RESULTS: M. genitalium LAMPs induced the expression of HO-1 mRNA and protein, the accumulation of ROS and the nuclear translocation of Nrf2 in placental trophoblast cells. NAC treatment inhibited LAMPs-induced HO-1 expression and Nrf2 nuclear translocation, and the transfection of Nrf2 siRNA significantly abrogated HO-1 expression. Furthermore, HO-1 siRNA and ZnPP treatment increased LAMPs-induced TNF-α and IL-1ß secretion, while the HO-1 agonist CoPP treatment further decreased their production. CONCLUSION: M. genitalium LAMPs could induce placental trophoblast cells to express HO-1 through ROS/Nrf2 pathways. Up-regulation of HO-1 negatively regulates excessive production of cytokines.

Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Mycoplasma fermentans inhibits the activity of cellular DNA topoisomerase I by activation of PARP1 and alters the efficacy of its anti-cancer inhibitor.

To understand the effects of the interaction between Mycoplasma and cells on the host cellular function, it is important to elucidate the influences of infection of cells with Mycoplasma on nuclear enzymes such as DNA Topoisomerase type I (Topo I). Human Topo I participates in DNA transaction processes and is the target of anti-cancer drugs, the camptothecins (CPTs). Here we investigated the mechanism by which infection of human tumor cells with Mycoplasma fermentans affects the activity and expression of cellular Topo I, and the anti-cancer efficacy of CPT. Human cancer cells were infected or treated with live or sonicated M. fermentans and the activity and expression of Topo I was determined. M. fermentans significantly reduced (by 80%) Topo I activity in the infected/treated tumor cells without affecting the level of Topo I protein. We demonstrate that this reduction in enzyme activity resulted from ADP-ribosylation of the Topo I protein by Poly-ADP-ribose polymerase (PARP-1). In addition, pERK was activated as a result of the induction of the MAPK signal transduction pathway by M. fermentans. Since PARP-1 was shown to be activated by pERK, we concluded that M. fermentans modified the cellular Topo I activity by activation of PARP-I via the induction of the MAPK signal transduction pathway. Moreover, the infection of tumor cells with M. fermentans diminished the inhibitory effect of CPT. The results of this study suggest that modification of Topo I activity by M. fermentans may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists.

Cathepsin L protects mice from mycoplasmal infection and is essential for airway lymphangiogenesis.

Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.

Expression and characterization of a Mycoplasma genitalium glycosyltransferase in membrane glycolipid biosynthesis: potential target against mycoplasma infections.

Mycoplasmas contain glycoglycerolipids in their plasma membrane as key structural components involved in bilayer properties and stability. A membrane-associated glycosyltransferase (GT), GT MG517, has been identified in Mycoplasma genitalium, which sequentially produces monoglycosyl- and diglycosyldiacylglycerols. When recombinantly expressed in Escherichia coli, the enzyme was functional in vivo and yielded membrane glycolipids from which Glcß1,6GlcßDAG was identified as the main product. A chaperone co-expression system and extraction with CHAPS detergent afforded soluble protein that was purified by affinity chromatography. GT MG517 transfers glucosyl and galactosyl residues from UDP-Glc and UDP-Gal to dioleoylglycerol (DOG) acceptor to form the corresponding ß-glycosyl-DOG, which then acts as acceptor to give ß-diglycosyl-DOG products. The enzyme (GT2 family) follows Michaelis-Menten kinetics. k(cat) is about 5-fold higher for UDP-Gal with either DOG or monoglucosyldioleoylglycerol acceptors, but it shows better binding for UDP-Glc than UDP-Gal, as reflected by the lower K(m), which results in similar k(cat)/K(m) values for both donors. Although sequentially adding glycosyl residues with ß-1,6 connectivity, the first glycosyltransferase activity (to DOG) is about 1 order of magnitude higher than the second (to monoglucosyldioleoylglycerol). Because the ratio between the non-bilayer-forming monoglycosyldiacylglycerols and the bilayer-prone diglycosyldiacylglycerols contributes to regulate the properties of the plasma membrane, both synthase activities are probably regulated. Dioleoylphosphatidylglycerol (anionic phospholipid) activates the enzyme, k(cat) linearly increasing with dioleoylphosphatidylglycerol concentration. GT MG517 is shown to be encoded by an essential gene, and the addition of GT inhibitors results in cell growth inhibition. It is proposed that glycolipid synthases are potential targets for drug discovery against infections by mycoplasmas.

Invasion of melanoma cells by Mycoplasma hyorhinis: enhancement by protease treatment.

Mycoplasma hyorhinis (strain MCLD) was recently isolated from a melanoma cell culture. Growth of MCLD was considerably improved by 24 serial passages in a modified Hayflick's mycoplasma medium. Transmission electron microscopy showed that MCLD exhibits a polymorphic appearance, with ovoid or elongated cells frequently harboring an electron-dense core at one of the poles. Adherence of M. hyorhinis to melanoma cells followed saturation kinetics. Furthermore, although M. hyorhinis has been considered to remain attached to the surface of the host cells, we show for the first time, qualitatively by confocal laser scanning microscopy and quantitatively by a gentamicin resistance assay, that MCLD is able to invade melanoma cells. The ingested mycoplasmas were randomly distributed in the cytoplasm, tending to concentrate near the plasma membrane. Both adherence to and invasion of melanoma cells by M. hyorhinis strain MCLD were dramatically enhanced by mild proteolytic digestion with proteinase K (2.5 microg/mg cell protein for 2.5 min at 37 degrees C) that affected the surface-exposed proteins of this organism, mainly the major 47-kDa lipoprotein. We suggest that the intracellular location of M. hyorhinis strain MCLD is a privileged niche, which may explain the survival of M. hyorhinis in tissue cultures. The enhanced binding to and invasion of melanoma cells by protease treatment may be due to either the activation or the enhanced exposure of an adhesin(s) on the mycoplasmal cell surface.

The importance of B-cells and ecto-5'nucleotidase in Mycoplasma fermentans infection and the relevance to rheumatoid arthritis.

The aim of this work was to discover if Mycoplasma fermentans, which is known to infect B cells, could be the cause of the raised ecto-5'-nucleotidase observed in the synovial fluid of rheumatoid arthritis patients. The ecto-5'-nucleotidase activity in the patients' serum has been shown to correlate with the erythrocyte sedimentation rate and DNA from the mycoplasma has been found in the synovial fluid. B lymphoblastoid cell lines were exposed to 16 strains of Mycoplasma fermentans and their ecto-5'-nucleotidase, CD73, was measured both biochemically and by mouse antibodies to human ecto 5'-nucleotidase using the fluorescence activated cell sorter. The type strain, PG 18, did not grow with the B cells. Some of the mycoplasma strains (9/15) increased the cellular ecto-5'-nucleotidase activity from twice to 17 fold, and usually showed 5'-nucleotidase activity themselves. At least one strain, M106, induced human 5'-nucleotidase on the normally 5'-nucleotidase negative Daudi and Raji Burkitt's lymphoma cell lines, and increased sevenfold the 5'-nucleotidase on the monocyte/macrophage cell line THP-1. Growing the cells in aged medium increased the level of mycoplasma infection. This mycoplasma-induced enzyme showed a conformational change and an increase in activity with a glycosylation change involving mannose groups. The other group of strains, mostly of respiratory or cell culture origin, usually did not have any 5'-nucleotidase of their own and decreased the B-cell enzyme activity by about half. Electron microscopy and flow cytometry showed that the strain M106 was filamentous and could be found inside the B-cells. The 5'-nucleotidase-inducing strains of M. fermentans may be important in the aetiology of rheumatoid arthritis.

Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis.

Mycoplasma alligatoris causes acute lethal cardiopulmonary disease of susceptible hosts. A survey of its genome implicated sialidase and hyaluronidase, synergistic regulators of hyaluronan receptor CD44-mediated signal transduction leading to apoptotic cell death, as virulence factors of M. alligatoris. In this study, after the existence of a CD44 homolog in alligators was established by immunolabeling primary pulmonary fibroblasts with monoclonal antibody IM7 against murine CD44, the sialidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was used to examine the effects of sialidase on fibroblast apoptosis following in vitro infection with M. alligatoris. While their CD44 expression remained constant, infected cells exhibited morphologic changes characteristic of apoptosis including decreased size, rounding, disordered alpha-tubulin, and nuclear disintegration compared to untreated controls. DANA was a potent, non-toxic inhibitor of the sialidase activity, equivalent to about 1mU of Clostridium perfringens Type VI sialidase, expressed by M. alligatoris in the inoculum. Although DANA did not measurably reduce the proportion of infected fibroblasts labeled by a specific ligand of activated caspases, co-incubation with DANA protected (P<0.01) fibroblasts in a concentration-dependent fashion from the M. alligatoris-induced trends toward increased apoptosis receptor CD95 expression, and increased 5-bromo-2'-deoxyuridine incorporation measured in a terminal dUTP nick end-labeling apoptosis assay. In contrast, incubation with 200-fold excess purified C. perfringens sialidase alone did not affect CD95 expression or chromatin integrity, or induce fibroblast apoptosis. From those observations we conclude that interaction of its sialidase with hyaluronidase or another virulence factor(s) is necessary to elicit the pro-apoptotic effects of M. alligatoris infection.

Effect of experimental genital mycoplasmosis on production of matrix metalloproteinases in membranes and amniotic fluid of Sprague-Dawley rats.

PROBLEM: Preterm, premature rupture of membranes (PPROM) is a dire pregnancy outcome that is frequently associated with infection by the genital mycoplasmas, Mycoplasma hominis, Ureaplasma parvum, and U. urealyticum. One potential mechanism by which these microorganisms may cause PPROM is by increasing the concentration of matrix metalloproteinases (MMPs) in the membranes and amniotic fluid. We tested this hypothesis in a well-defined model system of genital infection with M. pulmonis, a natural reproductive pathogen of rats. METHOD OF STUDY: Timed-pregnant, specific pathogen-free, Sprague-Dawley rats were infected with 10(7) CFU M. pulmonis at gestation day (gd) 14. Controls received an equivalent volume (100 microL) of sterile medium. At gd 18, rats were euthanized, and membranes and amniotic fluids were harvested and stored at -70 degrees C until analysis. Proteinase activity of amniotic fluid and membranes was resolved on discontinuous 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography gels. Band intensity was determined using a digital gel documentation system and the manufacturer's software (Kodak). RESULTS: Gelatinolytic activity associated with a band similar in molecular weight to ProMMP-9 (92 kDa, the inactive precursor of MMP-9) was significantly increased in amniotic fluids and membranes harvested from M. pulmonis-treated pups at gd 18 when compared with tissues harvested from control pups. Both ProMMP-9 and ProMMP-2 (72 kDa, the inactive precursor of MMP-2) were increased in infected animals at gd 21. CONCLUSION: Our study suggests that the genital mycoplasmas can increase MMP-9 production in vivo.

Vaginal microflora associated with bacterial vaginosis in nonpregnant women: reliability of sialidase detection.

OBJECTIVE: To determine the prevalence of Gardnerella vaginalis, anaerobic bacteria and Mycoplasma hominis in vaginal specimens of women with and without bacterial vaginosis (BV) as well as to determine the sensitivity and specificity of the direct sialidase assay of vaginal fluid as a rapid test for diagnosing this syndrome. METHODS: Vaginal cultures were obtained from 109 nonpregnant women (mean age 33 +/- 7.1 years), 47 of them with clinical signs of BV (BV+) and 62 of them without BV (BV-). In addition, we determined the vaginal sialidase activity in both groups, which may serve as a feature of this syndrome. RESULTS: Anaerobic bacteria were isolated in 91% and 18% of the BV+ and BV- groups, respectively (p < 0.001). Peptostreptococcus spp., Prevotella bivia and Porphyromonas spp. were strongly associated with BV. P. bivia and Prevotella spp. represented 44% of all the anaerobes isolated in the BV+ group. All the isolated P. bivia strains presented sialidase activity. G. vaginalis and M. hominis were isolated in 76% and 42% of the BV+ and 1% and 0% of the BV- women, respectively (p < 0.001). Mobiluncus morphotypes were observed in 34% of the BV+ and 0% of BV- women. Sensitivity, specificity, positive predictive value and negative predictive value of sialidase activity were 81%, 94%, 90% and 86%, respectively. CONCLUSIONS: Our data demonstrate a strong association between G. vaginalis, M. hominis, and P. bivia and BV. Sialidase activity and Gram stain of vaginal fluid represent accurate methods for diagnosing BV.

Mycoplasma infection can sensitize host cells to apoptosis through contribution of apoptotic-like endonuclease(s).

Mycoplasma infection may lead to various pathologies in a broad range of hosts. It has been shown that Mycoplasma may trigger cell death in cell cultures; however, the mechanism remains unknown. In the present paper we show that Mycoplasma infection of different lymphocyte and epithelial tumour cell lines leads to the inhibition of proliferation, and increased cell death, accompanied by DNA fragmentation and the morphological features of apoptosis. We also showed that this infection leads to an increased sensitivity of cells to various inducers of apoptosis targeting different signalling pathways. Finally, we show that increased apoptosis is associated with overexpression of an endonuclease produced by Mycoplasma. This endonuclease is recovered in the nuclear fraction of host cells, introduces mostly DSB and is active at neutral pH in the presence of divalent cations. Activation of this endonuclease is connected with limited proteolysis, which may be reproduced in vitro by snake venom serine proteinase.

Immune response against the L-lactate dehydrogenase of Mycoplasma hyopneumoniae in enzootic pneumonia of swine.

The L-lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae, formerly named protein P36, belongs to the predominant immunogenic proteins in pigs which were naturally or experimentally infected with M. hyopneumoniae. The antigenic reaction against M. hyopneumoniae LDH has been shown to be species specific. Recombinant M. hyopneumoniae LDH, which was genetically engineered to contain six histidine residues at its C-terminal end, was expressed in E. coli and purified to a high degree using Ni-chelate affinity chromatography. The genetically engineered LDH still showed the same biochemical activity and immunological specificity as the wild-type LDH and was used as an antigen for a M. hyopneumoniae LDH ELISA. Using this assay, we showed that pigs experimentally infected with M. hyopneumoniae raised antibodies against LDH in two steps. An early, relatively weak anti-LDH response was detected between 5 to 10 weeks post-infection when clinical signs and lung lesions occur. This first minor raise of anti-LDH antibodies occurred simultaneously with the strong appearance of antibodies against an antigen consisting of membrane proteins of M. hyopneumoniae prepared with Tween 20 extraction. A second, strong raise in anti-LDH antibodies was observed from the twelfth week after infection, at a time when the disease signs and the infectious agent disappeared. The high anti-LDH titer persisted until 21 weeks post-infection, in contrast to the antibody titer against the membrane proteins which started to decrease after its peak at 12 weeks post-infection. A LDH-ELISA may also be useful for detecting past infections.

The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures.

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filtration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.

A novel ornithine transcarbamylase present in mycoplasma-infected myeloma cells.

A myeloma cell line (KHM-4) from a patient with multiple myeloma and idiopathic hyperammonemia, and another myeloma cell line (RPMI8226) were seen to have activity to form ammonia from arginine. High activity of ornithine transcarbamylase (OTC), a hepatic urea cycle enzyme, was detected in these cell lines. OTC of these cells was much more heat-stable than the liver enzyme, and did not cross-react with an antibody against the liver enzyme. As the OTC activity was also detected in the culture medium of the myeloma cells and because the activity was markedly decreased by the antimycoplasma drug MC-210, the OTC activity was assumed to be associated with mycoplasma infection. Polymerase chain reaction, using degenerate oligonucleotide mixtures corresponding to the two highly conserved sequences of OTC, amplified a DNA sequence that apparently encodes a portion (about 67% in length) of mycoplasma OTC. The predicted amino acid sequence of the mycoplasma enzyme was 33-47% identical with those of the enzymes of bacteria, yeast and mammals.

Increasing values of serum acid phosphatase in a child with Mycoplasma pneumoniae-associated hemophagocytic histiocytic syndrome.

We describe a 3 1/2-year-old boy with disseminated histiocytic disease probably induced by Mycoplasma pneumoniae. In this patient, acid phosphatase was elevated in serum and was also detected histochemically in the infiltrating histiocytes. The serum acid phosphatase levels increased as his histiocytosis progressed, apparently mirroring the activity of the disease. This observation suggests that serum acid phosphatase levels should be evaluated further to determine whether they will be a useful indicator of disease in children with different histiocytosis syndromes.

Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections.

Neuropeptides such as substance P are implicated in inflammation mediated by sensory nerves (neurogenic inflammation), but the roles in disease of these peptides and the peptidases that degrade them are not understood. It is well established that inflammation is a prominent feature of several airway diseases, including viral infections, asthma, bronchitis, and cystic fibrosis. These diseases are characterized by cough, airway edema, and abnormal secretory and bronchoconstrictor responses, all of which can be elicited by substance P. The effects of substance P and other peptides that may be involved in inflammation are decreased by endogenous neutral endopeptidase (NEP; also called enkephalinase, EC 3.4.24.11), which is a peptidase that degrades substance P and other peptides. In the present study, we report that rats with histories of infections caused by common respiratory tract pathogens (parainfluenza virus type 1, rat corona-virus, and Mycoplasma pulmonis) not only have greater susceptibility to neurogenic inflammatory responses than do pathogen-free rats but also have a lower activity of NEP in the trachea. This reduction in NEP activity may cause the increased susceptibility to neurogenic inflammation by allowing higher concentrations of substance P to reach tachykinin receptors in the trachea. Thus decreased NEP activity may exacerbate some of the pathological responses in animals with respiratory tract infections.

Decrease in catalase activity of cultured cells by Mycoplasma gallisepticum infection.

The effect of Mycoplasma gallisepticum infection on the host cell catalase activity was histochemically examined in cultured chicken embryo fibroblasts (CEF) and kidney cells. The activity in normal cells was detected as fine, brown granules in the cytoplasm, which appeared ultrastructurally to correspond to anucleoid microbodies. By infecting cultured cells with a CEF-passaged strain of M. gallisepticum, the catalase-positive granules clearly decreased in amount, whereas the UV light-killed mycoplasma and the original strain failed to decrease the granules. The cell-passaged strain was able to induce cytopathic effects and this appeared to be due to its enhanced adherent ability as compared with the original strain. These findings suggest that attachment of viable organisms to cells is crucial to decrease the catalase activity and that the decreased activity may be an important process for the subsequent development of cytopathic effects.

Leukocyte esterase activity in the rapid detection of urinary tract and lower genital tract infections in obstetric patients.

Infections of the vagina and urinary tract are important problems for the obstetrician. Examination of the vaginal discharge and urine for the presence of leukocytes is an important part of the evaluation for vaginitis and urinary tract infections. Neutrophils contain several esterases that are not present in serum, urine, or vaginal secretions. These esterases are not influenced by bacteria, commonly used drugs, or variable compositions of urine or vaginal secretions. A prospective study was performed to assess the sensitivity and specificity of leukocyte esterase activity as measured by dipstick (Chemstrip 9, Biodynamics) for the prediction of vaginitis and urinary tract infections during pregnancy. Results were compared with those obtained from potassium hydroxide smears, wet preps, and urine cultures. The vaginal discharge and urine of 65 patients was tested for leukocyte esterase activity on their initial OB visit. Leukocyte esterase was 100% sensitive and 100% specific for detecting urinary tract infections. It was 100% sensitive and 90% specific for predicting vaginal infections. Trichomonas infections accounted for the positive leukocyte esterase results when the urine culture was negative. On the basis of this study we believe that leukocyte esterase activity is sufficiently sensitive and specific to permit use of this test as a rapid and inexpensive screening procedure for vaginitis and urinary tract infections.

Influence of Mycoplasma arginini infection on the induction of aryl hydrocarbon hydroxylase by TCDD in rat hepatoma cell cultures.

Mycoplasma arginini was eliminated from a rat hepatoma cell line (H-4-II-E) by plating at low cell density and treatment with chlortetracycline (250 micrograms/ml), kanamycin (250 micrograms/ml), tylosin (100 micrograms/ml), 3% M. arginini antiserum and 5% fresh guinea-pig serum. The induction of AHH activity in the cell culture was measured in response to increasing concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The ED50 values (estimated doses that produce 50% maximum enzyme induction) were calculated to be 0.256, 0.452 and 0.344 pmol TCDD/plate for original, mycoplasma-free and reinfected cells, respectively. Although the absence of M. arginini in the rat hepatoma cell line makes the cells slightly less responsive to AHH induction by TCDD, this decrease does not detract from the use of the method to screen food extracts and environmental samples for the presence of certain toxic planar organic compounds.