Small Animal Critical Care Medicine, 1e
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Denny and Steven J. Simon Turner and C. Sharp and Simon J. Scoott and William H. Miller Jr. The cell lysates were subject to immunoblot analysis as in C. Cells and the overlying medium supernatant were separated and utilized for evaluation of PXDN-mediated bactericidal killing. The control group contained untreated AECs and P. Wild-type mice that survived acute infection at 48 h appeared to recover completely and remained healthy for several days. PBS treatment did not result in death in either strain of mice, and bacteria were absent in the lungs of these mice Fig 5A and 5B.
Intratracheal injection with sublethal dose of P. The infected lungs of PXDN-deficient mice revealed more severe tissue injury and neutrophil infiltration, while uninfected lungs showed normal structure Fig 5E. Together, these studies provide compelling data to support a critical host defense function of PXDN, specifically against GN bacterial pneumonia.
A Decrease of relative survival rates. B Lung Bacterial burden.capamefrawi.tk/mas-de-40-gratis.php
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Mice were sacrificed at 20 hours; lungs were aseptically removed, weighed, and homogenized in PBS. Lung tissue suspension was serially diluted and plated on LB agar plates. C and D Tissue burden on bacterial infection with sublethal dose was carried out by intratracheally infecting the mice with 3 x 10 6 of P.
After 20 h, lung, liver and spleen were taken as in B for detection of bacteria. Mice were infected as in C and the lungs were harvested at 20 h for preparation of staining. WT mouse, uninfected; b, WT mouse, infected; c. The lung is a uniquely vulnerable organ with a very thin, delicate epithelial lining, abundant blood flow, and a vast surface area.
The lung resides at the interface of the body and environmental exposures to inhaled or aspirated pathogens. Thus, the lung is an important organ in host defense. Multiple layers of defense in the normal lung are involved in innate immune functions. Loss of one or more of these host defense mechanisms increases the susceptibility of the lung to infections. PXDN is a newly identified hPx, an enzyme family that plays an important role in host defense. Its physiological function is largely unknown, although recent studies implicate this gene in basement membrane synthesis [ 28 , 29 ].
The present study, for the first time, identifies PXDN as a novel host defense enzyme in the lung with selectively for recognizing and directly killing GN bacteria. An enzyme containing the molecular structure of both pattern-recognition domain and scavenger domain suggests evolutionary conservation of a dual-function protein capable of both pathogen recognition and killing, expanding our current view of innate immunity.
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The original theory of innate immune pattern recognition is based on the interaction between host PRRs and specific PAMPs, and the activation of downstream signaling events and host defense mechanisms [ 4 , 5 ]. On the other hand, many effectors including complement system, antimicrobial peptides, and lysozymes may bind to important bacterial molecules and directly kill pathogens. For example, neutrophils possess bactericidal permeability-increasing protein BPI in their azurophilic granules.
Peptidoglycan recognition proteins PGRPs are innate immune molecules present in insects, mollusks, echinoderms, and vertebrates. Mammals have four PGRPs. The three remaining PGRPs kill bacteria by interacting with cell wall peptidoglycan [ 31 ].
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PXDN is found in circulating plasma at a concentration of 1. The high expression of PXDN in alveolar epithelium and in bronchoalveolar lavage fluid suggests that this enzyme may mediate critical host defense functions and mucosal immunity. The ability of PXDN to directly bind GN bacterial pathogens allows for more targeted activation and killing without collateral damage to surrounding tissues. The concept of targeted killing is further supported by the finding that LPS induces PXDN activation, a phenomenon has not been reported for other members of the hPx family.
High incidence, infection severity and increasing resistance characterize P. In summary, PXDN is a novel host defense enzyme in the lung with dual function in pathogen recognition and killing. Further studies are required to determine its role in systemic immunity. The unique structural and functional characteristic of PXDN expands our current understanding of mucosal innate immunity, and has important implications for novel therapeutic strategies.
Unless otherwise stated, mice were fed with normal chow diet. The protocol of animal study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham with approval number The mixture was incubated at RT for 1 h. Bacteria were spun down at x g for 5 min. The cells were washed twice by 5 x initiating volume of PBS.
Protein bands were visualized by chemiluminescence. Negative controls contained only PXDN or bacteria. In some experiments, LPS-deficient E. LPS-deficient E. Control groups were cells or plasma alone.
In some experiments, lipid A Sigma-Aldrich Cat. L was used. The mixture was shacked at RT for 15 min. Then 2 x 10 8 live E. Bacterial pellets were obtained and subject to immunoblot analysis. Full kinetics was carried out by flowing a serial concentration range from 0. All measurements were conducted in triplicate.
Rate constants of SPR were calculated as following. Reaction mixture was incubated at RT for 30 min. After 30 min, absorbance at nm was recorded. Other halide anions bromide, iodide and thiocyanate in addition to Cl - were used as indicated. GP bacteria were also utilized in some bacterial killing experiments. Colonies were counted.
Chemiluminescent dye L is a sensitive substrate for measuring heme-containing peroxidase activity. It generates chemiluminescence once oxidation. Chemiluminescent light at nm was immediately recorded by a luminometer Molecular Devices, Sunnyvale, CA. In brief, 4—5 mice were euthanized with CO 2. Blood was exsanguinated by clipping abdominal aorta. Trachea and lungs were carefully exposed and lungs were perfused through puncture of right ventricle with 10 mL of sterile PBS until lungs clear of blood.
Lungs were carefully removed and incubated in dispase II solution for 45 min. Lung tissue was minced with scissors until the consistency of jelly. BD, The media containing unbound cells were carefully removed and placed into culture dish. Next day, the media containing non-adherent cells were transferred into 50 mL-conical tube. The remaining adherent cells were fibroblast. The cell pellet was re-suspended in complete media.
Cells were placed into fibrinogen coated plates.
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All steps were carried out aseptically. Human plasma and purified MPO Cat. Cells were serum-starved for 16 hrs. Cells and medium were separated for evaluation of the bacterial killing, respectively. Inoculums for mouse infections will be prepared as previously described with modification [ 33 ].
This stock will be diluted in sterile PBS to give an appropriate titer. Mice were allowed to recover for 30—60 min prior to being returned to the cage. The tissue was weighed and homogenized for bacteria clearance analysis. Homogenized tissue was washed with 4 mL sterile PBS. The bacterial resuspension was serial dilution. Bacterial colonies were counted for analysis. Experiment was carried out as in Fig 1D.
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Samples were subjected to immunoblotting using anti- PXDN or anti-ceruloplasmin antibodies. Experiments were similar to Fig 3B and 3C. Indicated number of bacteria was added into TMB solution. Indicated number of E. The experiment was similar to that in Fig 3D. The CFUs were counted, and relative survival rates were calculated. Photographs of petri dishes were taken. Data are representatives of three plates. Two lots of recombinant PXDN were used. We thank Drs. Joao A. Harvey Pough. Lester Late. Zitelli MD. Gahart RN.
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