Agar Beads Research Articles

Immobilization of Brevibacterium Cells for the production of l-glutamic acid

Studies were performed to elucidate the optimal conditions (gel concentration, bead size, initial biomass, storage period) for immobilization of growing cells of Brevibacterium sp. (DSM 20411) in alginate and agar gels and to evaluate the immobilized biocatalyst in batch mode, and continuous mode in a packedcolumn bioreactor, for l-glutamic acid production. The entrapped cells were also cultivated for fwenty five days in five repeated batches (each batch lasted for five days). Maximum l-glutamic acid production in batch and repeated-batch modes was 7.4 and 8.7 mglml, respectively, with alginate beads, and 11.8 and 13.3mg/ml, respectively, with agar beads. Under optimized conditions (flow rate, initial cell loading), l-glutamate productivity was maintained within a range 10–11 mg/ml for two weeks, in continuous mode, in a packed column bioreactor with agarimmobilized cells.

Relevance of rheological properties of gel beads for their mechanical stability in bioreactors

The mechanical stability of biocatalyst particles in bioreactors is of crucial importance for applications of immobilized-cell technology in bioconversions. The common methods for evaluation of the strength of polymer beads (mostly force-to-fracture or tensile tests) are, however, not yet proven to be relevant for the assessment of their mechanical stability in bioreactors. Therefore, we tested fracture properties of gel materials and investigated their relevance for abrasion in bioreactors. Abrasion of gel beads was assumed to be a continuous fracturing of the bead surface. At first, three rheological properties were considered: stress at fracture; strain at fracture; and the total fracture energy. If stress at fracture is the most important property, beads having a similar fracture energy, but a smaller stress at fracture, would abrade faster in a bioreactor than beads with a larger stress at fracture; if fracture energy the determining factor, beads that require less energy to fracture would abrade faster than those having a larger fracture energy for the same fracture stress. To determine this, beads of kappa-carrageenan and agar (at two different polymer concentrations) were tested for abrasion in four identical bubble columns under the same operating conditions. Agar beads were expected to abrade faster than those of carrageenan because agar had either a lower stress at fracture or a lower fracture energy. However, no correlation between fracture properties and abrasion rate was found in any of the combinations tested. Carrageenan beads abraded faster than those of agar in all combinations. Furthermore, both the stress and strain at fracture of agar and carrageenan beads decreased during the run and those of carrageenan decreased faster, suggesting that the gels are liable to fatigue in different ways. This hypothesis was confirmed by oscillating experiments in which gel samples were subjected to repeated compressions below their fracture levels. Their resistance to compression clearly decreased with the number of oscillations. Fatigue is probably related to the development of microcracks and microfracture propagation within the material. We concluded that: (a) the use of tests based on bead rupture do not provide relevant information on the mechanical stability of gel beads to abrasion; and (b) abrasion of polymer beads is likely to be related to fatigue of the gel materials. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 517-529, 1997.

Relevance of rheological properties of gel beads for their mechanical stability in bioreactors

The mechanical stability of biocatalyst particles in bioreactors is of crucial importance for applications of immobilized-cell technology in bioconversions. The common methods for evaluation of the strength of polymer beads (mostly force-to-fracture or tensile tests) are, however, not yet proven to be relevant for the assessment of their mechanical stability in bioreactors. Therefore, we tested fracture properties of gel materials and investigated their relevance for abrasion in bioreactors. Abrasion of gel beads was assumed to be a continuous fracturing of the bead surface. At first, three rheological properties were considered: stress at fracture; strain at fracture; and the total fracture energy. If stress at fracture is the most important property, beads having a similar fracture energy, but a smaller stress at fracture, would abrade faster in a bioreactor than beads with a larger stress at fracture; if fracture energy the determining factor, beads that require less energy to fracture would abrade faster than those having a larger fracture energy for the same fracture stress. To determine this, beads of κ-carrageenan and agar (at two different polymer concentrations) were tested for abrasion in four identical bubble columns under the same operating conditions. Agar beads were expected to abrade faster than those of carrageenan because agar had either a lower stress at fracture or a lower fracture energy. However, no correlation between fracture properties and abrasion rate was found in any of the combinations tested. Carrageenan beads abraded faster than those of agar in all combinations. Furthermore, both the stress and strain at fracture of agar and carrageenan beads decreased during the run and those of carrageenan decreased faster, suggesting that the gels are liable to fatigue in different ways. This hypothesis was confirmed by oscillating experiments in which gel samples were subjected to repeated compressions below their fracture levels. Their resistance to compression clearly decreased with the number of oscillations. Fatigue is probably related to the development of microcracks and microfracture propagation within the material. We concluded that: (a) the use of tests based on bead rupture do not provide relevant information on the mechanical stability of gel beads to abrasion; and (b) abrasion of polymer beads is likely to be related to fatigue of the gel materials. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 517–529, 1997.

Rock phosphate solubilization by immobilized cells of Enterobacter sp. in fermentation and soil conditions

An Enterobacter sp., a phosphate-solubilizing bacterium, was immobilized in agar. Various amounts of the immobilized bioparticles were applied in a repeated-batch fermentation process in order to solubilize Venezuelan rock phosphate. A total production of 388 mg and 403 mg soluble P/l was obtained for 2.0 g rock phosphate/l in flasks containing 10 ml and 20 ml agar beads, respectively, that was significantly higher compared to a free-cell single-batch control. Whole and destroyed immobilized bioparticles were introduced into a soil enriched with rock phosphate to improve the growth of onion plants. Compared to the control supplemented with a free-cell bacterial suspension, the results showed at least equal plant growth of 80 mg/pot and shoot P concentration of 82 μg/plant when 10 destroyed beads were applied per pot.

Electron microscopic studies on immobilized growing Chlamydomonas reinhardtii cells

Electron microscopic observations of Chlamydomonas reinhardtii cells immobilized in agar beads revealed that many colonies are assembled over the gel surface and are enlarged during growth. When the microalga reached the stationary phase of growth, the chlorophyll content in the gel beads remained constant, but the gel surface showed microinterstices that allow cell release to the culture medium. The viability was maintained by the cells; thus, this system could be interesting in applied processes operating continuously.

Role of coagulase in a murine model of hematogenous pulmonary infection induced by intravenous injection of Staphylococcus aureus enmeshed in agar beads.

We describe a novel mouse model of acute staphylococcal pneumonia induced by intravenous injection of Staphylococcus aureus enmeshed in agar beads. For comparison, we also used various strains of bacteria, including three strains of S. aureus, two strains of Staphylococcus epidermidis, one strain of Streptococcus pyogenes, three strains of Pseudomonas aeruginosa, and one strain of Klebsiella pneumoniae. All except two strains of S. aureus were cleared rapidly from the lungs. When S. aureus NUMR1 enmeshed in agar beads was injected intravenously, the organisms concentrated and remained in the lung for a period longer than several weeks. Multiple lung abscesses were evident macroscopically, and histological examination of the infected lung showed multiple lung abscesses around the pulmonary arterioles, consisting of bacterial colonies encircled with fibrin filaments and surrounded by inflammatory cells of neutrophils and macrophages. When 14 strains of clinically isolated S. aureus were injected intravenously, the number of bacteria recovered from the lung tissue 7 days after infection correlated with the titer of staphylocoagulase (P < 0.01) but not with the titer of clumping factor. Injection of coagulase-deficient mutant strain DU5843 was associated with a markedly reduced number of viable bacteria isolated from the lung, compared with its coagulase-positive parental strain DU5789. Our results suggest that coagulase may play a role in the development of blood-borne staphylococcal pneumonia in our model. Our animal model is simple and reproducible and resembles blood-borne staphylococcal pneumonia in humans, and it could be useful for investigating the pathogenicity or treatment of staphylococcal pulmonary infection, including infections with methicillin-resistant S. aureus.

Effect of 6-fluoro-8-methoxy quinolone (AM-1155) against chronic airway infection with Pseudomonas aeruginosa in a rat model.

We studied the effect of AM-1155, a newly developed quinolone, against chronic airway infection with Pseudomonas aeruginosa using a previously described rat model. AM-1155 (25 mg/kg) or ciprofloxacin (25 mg/kg) or saline (controls) were injected s.c. for 14 days (from day 4 to day 17) after the inoculation of agar beads containing P. aeruginosa. The number of viable cells of intrapulmonary P. aeruginosa, histological findings of the lungs and immunoglobulin levels of serum and bronchoalveolar lavage fluid were examined in rats 11 and 18 days after the treatment. The findings indicated that the number of viable cells of P. aeruginosa in lungs was significantly decreased in the AM-1155- or ciprofloxacin-treated group compared with the non-treated control group. Histological examination in the non-treated control group showed hyperplasia of bronchus-associated lymphoid tissue as well as cellular infiltration in airways, but not prominently in the AM-1155- or ciprofloxacin-treated group. The IgG and IgA levels in serum and bronchoalveolar lavage fluid were significantly lower in the AM-1155- and ciprofloxacin-treated groups than in the control group. These in-vivo effects of AM-1155 were comparable to those of ciprofloxacin. These findings suggest that treatment with AM-1155 and ciprofloxacin suppressed excessive immune responses, preventing progression of airway damage in the chronic infectious state.

A study of bronchus-associated lymphoid tissue in a rat model of chronic pulmonary infection with Pseudomonas aeruginosa.

The immunologic pathogenesis of conditions characterized by chronic pulmonary infections, such as diffuse panbronchiolitis and those associated with cystic fibrosis, had not been fully clarified. Organized lymphoid tissue along the airway has been termed bronchus-associated lymphoid tissue (BALT), and hyperplasia of BALT is frequently observed in chronic pulmonary infections in humans. To investigate the role of BALT, we intratracheally inoculated rats with Pseudomonas aeruginosa (PA) enmeshed in agar beads according to the method of Cash et al., thereby establishing a chronic pulmonary infection model. Histopathological examination of tissue from this rat model revealed the accumulation of lymphocytes and foamy cells around bronchioles. This finding corresponds to chronic bronchiolitis in humans. Hyperplasia of BALT was also observed. Immunohistochemical examination demonstrated that Ia+ cells, helper T cells, surface IgM-positive (sIgM+) cells and sIgA+ cells had gradually increased in BALT and the walls of peripheral airways during the period from day 4 to 7. The anti-PA IgA antibody titer in bronchoalveolar lavage fluid (BALF) was also elevated during this period. After day 21, non-helper T cells became predominant in tissue sections, and the numbers of various immunoglobulin-positive cells as well as the anti-PA IgA antibody titer in BALF were reduced. Histological examination revealed that the inflammatory findings had also diminished. The time course of changes in the various immune cells in BALT and the walls of peripheral airways, paralleled the reductions in anti-PA IgA antibody titers in BALF. Our findings suggest that hyperplastic BALT may be one source of the Ig producing cells which play an important role in the local immune response characteristic of chronic pulmonary infections.

Efficacy of a subcutaneously administered, ultraviolet light-killed Pasteurella haemolytica A1-containing vaccine against transthoracic challenge exposure in goats

Abstract Objective To determine the effectiveness of Pasteurella haemolytica biovar A, serovar 1 (Ph A1) killed by UV light and incorporated with an oil adjuvant or carriers. Animals 40 weanling male Spanish goats. Procedure Goats were randomly allotted to 1 of 6 treatment groups: 4 Ph A1 bacterins (agar beads, polyacrylate beads [PA], phosphate-buffered saline solution, Freund's incomplete adjuvant), live Ph A1 with polyacrylate beads (LiPhPA), and polyacrylate beads (UnVac). Each of 4 Ph A1 vaccines was administered SC twice, 21 days apart, to 1 of 4 groups; another group received only PA beads SC, and the last group received live Ph A1 with PA beads by transthoracic injection into the left lung. 14 days after the second vaccination, all goats were challenge exposed with live Ph A1 by transthoracic injection into the right lung, and 4 days later, all goats were euthanatized and necropsied. Results Mean volume of consolidated right lung tissue was 1.02 cm3 for the LiPhPA group, 168.1 cm3 for the UnVac group, 2.3 cm3 for the Freund's incomplete adjuvant bacterin group, 5.53 cm3 for the PA bacterin group, 9.01 cm3 for the agar beads bacterin group, and 7.51 cm3 for the phosphate-buffered saline solution bacterin group. Mean volume of consolidated lung tissue was significantly different between the UnVac group and the other 5 groups. Conclusion The LiPhPA group and 4 bacterin groups developed protective immunity against live Ph A1 challenge exposure. Clinical Relevance An SC administered, UV light- killed Ph A1 bacterin induced protective immunity equal to that induced by virulent live Ph A1 injected into the target organ, the lung. (Am J Vet Res 1996;57:1168-1174)

Immobilization of prostaglandin synthetase by hydrophobic adsorption.

In this article, the immobilization of prostaglandin synthetase on n-alkyl or aryl amino-agar beads by hydrophobic adsorption is reported. The effects of different hydrophobic groups in the agar beads, pH of buffer, concentration of salts on the adsorption of prostaglandin synthetase, and the properties of immobilized prostaglandin synthetase were also studied. The results showed that 20-35 mg of microsome containing PG synthetase (protein content 8-15 mg) could be adsorbed on each gram of n-dodecylamino-agar beads after suction drying the gel in the buffer of pH 5.5 (containing 0.5 mol/L KCl), 0.1 mol/L citric-phosphate at 4 degrees C. The remaining immobilized enzyme activity was over 80%. The optimum pH of immobilized PG synthetase is 8.0, similar to that of the native enzymes. The thermostability of immobilized PG synthetase in the buffer containing 0.5 mol/L KCl was increased. Immobilized PG synthetase was used as a catalyst of synthesis of prostaglandin E1. The preservation of activity after 10 working cycles was 86.2%.

Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection.

Despite controversies associated with forms and value of antibiotic therapy for cystic fibrosis patients, antibiotherapy remains a cornerstone in the management of those patients. Locally administered liposome-encapsulated antibiotics may offer advantages over free antibiotics, including sustained concentration of the antibiotic, minimal systemic absorption, reduced toxicity, and increased efficacy. We evaluated the efficacy of free and encapsulated tobramycin in fluid and rigid liposomal formulations administered to rats chronically infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(5) CFU of a mucoid variant of P. aeruginosa PA 508 prepared in agar beads. Antibiotic treatments were given intratracheally at time intervals of 16 h. After the last treatment, lung bacterial counts were determined and tobramycin levels in the lungs and kidneys were evaluated by high-performance liquid chromatographic analysis and microbiological assay. Two independent experiments showed that animals treated with encapsulated tobramycin in fluid liposomes had a number of CFU less than the minimal CFU number required to be statistically acceptable compared with > or = 10(6) CFU per pair of lungs for animals treated with encapsulated tobramycin in rigid liposomes, free antibiotic, or liposomes without tobramycin. Tobramycin measured in the lungs at 16 h after the last treatment following the administration of encapsulated antibiotic was still active, and its concentration was > or = 27 micrograms/mg of tissue. Low levels of tobramycin were detected in the kidneys (0.59 to 0.87 micrograms/mg of tissue) after the administration of encapsulated antibiotic, while 5.31 micrograms/mg of tissue was detected in the kidneys following the administration of free antibiotic. These results suggest that the local administration of fluid liposomes with encapsulated tobramycin could greatly improve the management of chronic pulmonary infection in cystic fibrosis patients.

Characterization of bovine pulmonary and serum antibody responses after parenteral or intrapulmonary vaccination with live Pasteurella haemolytica

Pulmonary and serum antibody responses were evaluated in eight calves vaccinated [four intrapulmonary-right diaphragmatic lobe (IP) and four subcutaneous (SC)] with Pasteurella haemolytica A1 (Ph-1) impregnated agar beads and eight respective sham-vaccinated calves. Experimental and sham groups were challenged in both diaphragmatic lobes with Ph-1 34–37 d after vaccination (DAV) and necropsied 6 d after challenge (DAC; 40–43 DAV). IgG antibodies contained in fluids from the diaphragmatic lobes of vaccinated calves had different patterns of antigen specificity compared with IgG antibodies in analogous sera. Using ELISA, anti-Ph-1 IgA and IgG antibody concentrations were significantly higher ( P < 0.05) in lung lavage fluids from the IP group before and after challenge compared to the SC and sham groups. The IP and SC groups developed IgA, IgG and IgM antibody titers in nonvaccinated lung lobes after vaccination and challenge. The IP and SC groups exhibited significantly ( P < 0.05) smaller pulmonary lesions than the sham groups and pulmonary IgG and IgA antibodies were associated with increased protection.

Role of tumor necrosis factor alpha in innate resistance to mouse pulmonary infection with Pseudomonas aeruginosa

In the present study, we have investigated the mechanisms underlying mouse resistance to endobronchial infection with Pseudomonas aeruginosa enmeshed in agar beads. This was done by monitoring macrophage activation-associated gene expression in lung and alveolar cells harvested from resistant (BALB/c) and susceptible (DBA/2, C57BL/6, and A/J) strains of mice over the course of infection with P. aeruginosa. Interleukin-1 alpha, interleukin-1 beta, macrophage inflammatory protein-1 alpha, JE, and tumor necrosis factor alpha (TNF-alpha) mRNA expression levels were up-regulated in all strains of mice during the early phase of the infection. The level of TNF-alpha mRNA expression was increased to a greater extent in resistant BALB/c mice than in susceptible DBA/2, C57BL/6, and A/J strains of mice. This observation paralleled a higher secretion of TNF-alpha into the alveolar space of BALB/c mice at 3 and 6 h postinfection. The concentration of TNF-alpha released in alveoli returned to basal levels within 24 h of infection in mice of all strains, even though the TNF-alpha mRNA expression remained high until 3 days after infection. In vivo treatments with either anti-murine TNF-alpha monoclonal antibodies or with aminoguanidine significantly increased the number of P. aeruginosa bacteria detected in the lungs of resistant mice at 3 days postinfection. Overall, these findings indicate that both TNF-alpha and nitric oxide exert a protective role in response to pulmonary infection with P. aeruginosa.

Relevance of animal models for chronic bacterial airway infections in humans.

Animal models of chronic bacterial airway infections are limited in relevance to human infection, not only because of limitations present in all animal models (e.g., abnormal sequence of pathologic events, artificial imposition of infection involving large inocula, dissimilar host pharmacokinetic attributes), but especially because underlying disease (chronic obstructive pulmonary disease, or COPD) is usually absent. A major problem in establishing chronic lung infection models is the rapid clearance of bacteria instilled into the drugs; this can be overcome by encasing bacteria into agar beads to restrict phagocytosis. Despite these limitations, animal models based upon agar bead-encased inocula have provided persistent infections resulting in lung pathology and high humoral immune responses similar to that observed in patients with COPD. Such models have illuminated some aspects of the virulence of the bacteria, the involvement of bacterial exoenzymes in promoting lung damage, the potential of vaccination to restrict infectious exacerbations, and the activity of subminimal inhibitory concentrations of antibiotics in reducing lung damage by inhibiting exoenzyme release. Animal models have also shown that anti-inflammatory therapy may be effective in reducing inflammation-associated lung damage. Despite the limitations of chronic lung infection models, they may provide insight into pathologic events and positive therapeutic intervention for lung infections of patients with COPD.

Synthetic peptides representing two protective, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa elicit whole-cell-reactive antibodies that are functionally pseudomonad specific.

Peptide 9 (TDAYNQKLSERRAN) and peptide 10 (NATAEGRAINRRVE) represent surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Rats immunized with four intramuscular inoculations on days 0, 14, 28, and 42 with either peptide 9 or peptide 10 conjugated to keyhole limpet hemocyanin were afforded protection against pulmonary lesions when examined 7 days subsequent to challenge (day 56) via intratracheal inoculation of P. aeruginosa-containing agar beads. Peptide 9 shares considerable homology with other OmpA-related outer membrane proteins in various bacteria, whereas peptide 10 displays little homology with these other proteins. Antisera directed to peptide 9 reacted weakly with cell envelope proteins from the various other OmpA-associated bacteria upon immunoblot analysis. However, antisera directed to peptide 10 reacted only with Neisseria gonorrhoeae cell envelope proteins upon immunoblot analysis. Antisera to both peptides 9 and 10 reacted at minimal titers with whole cells of the various other bacteria in a whole-cell enzyme-linked immunosorbent assay (ELISA) but antisera to each of the peptides reacted at high titers when various strains of P. aeruginosa were used as the ELISA antigen. Antibodies to peptides 9 and 10 were protective, reactive to all strain of P. aeruginosa tested except for a protein F-deficient mutant, and functionally specific against pseudomonads.

Efficacy of a capsule preparation and ultraviolet-killed Pasteurella haemolytica A1 vaccine in goats

Weanling male Spanish goats ( n = 38) were randomly allotted to six treatment groups. Eight goats were positive controls; six goats were negative controls; six were given 2 transthoracic injections into the lung 21 d apart with 54 mg of purified Ph1 capsular polysaccharide (CPS) in agar beads; six had ultraviolet-killed Ph1 (1.4 × 10 9 CPU/ml) mixed with agar beads (Ph1-O); six had ultraviolet-killed Ph1 (8 × 10 9 CFU/ml) impregnated inside agar beads (Ph1-I); and six goats were given 2 subcutaneous injections 21 d apart with ultraviolet-killed Ph1 (8 × 10 9 CFU/ml) impregnated into agar beads (Ph1-I-SC). Thirteen d after the second injection all goats were challenge exposed to live Ph1 (1.93 × 10 8 CFU/ml) by transthoracic injection into the right lung, and 4 d later all goats were euthanatized and necropsied. Mean volume of consolidated lung tissue was: for the negative control group, 150.83 cm 3; CPS group, 113.98 cm 3; positive control group, 0.06 cm 3; Ph1-O group, 11.68 cm 3; Ph1-I group, 11.24 cm 3; and Ph1-I-SC group, 33.47 cm 3. There was a significant difference ( P < 0.04) in lesion size among treatment groups. Mean values for treatment groups, positive control, Ph1-O, and Ph1-I were clustered together and all were considered immune. The Ph1-I-SC group was considered partially immune, and the NC and CPS groups were considered non-immune. All of the negative control goats died on d 35, five of the capsular group died by d 36. The transthoracicly injected, ultraviolet-irradiated whole cell bacterin, whether outside or inside the beads, was considered an excellent bacterin which induced solid immunity to a severe challenge exposure.

Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa.

The pulmonary residence time of free and liposome-encapsulated tobramycin was studied with uninfected rats and rats infected with Pseudomonas aeruginosa. Chronic infection in lungs was established by intratracheal administration of 10(8) CFU of P. aeruginosa PA 508 prepared in agar beads. After 3 days, a single dose (300 micrograms) of free or liposome-encapsulated tobramycin was given intratracheally to both infected and uninfected rats. At various time intervals (0.25 to 16 h) after drug instillations, the remaining tobramycin was evaluated in blood, lungs, and kidneys by a microbiological assay. Intratracheal instillation of liposome-encapsulated tobramycin resulted in high and sustained levels of tobramycin in lungs of uninfected and infected rats over the 16-h period studied; however, the tobramycin levels were two times higher in uninfected rats. There was no tobramycin detected in the blood or kidneys from these animals. In contrast, the intratracheally instilled free tobramycin was cleared within 3 and 1 h from the lungs of uninfected and infected animals, respectively. These data suggest that the encapsulation of tobramycin in liposomes can result in a significant increase of its residence time within lungs. This study also shows that pulmonary infection was associated with a lowering of tobramycin levels in lungs.

Preservation of Pulmonary Function by an Outer Membrane Protein F Vaccine: A Study in Rats With Chronic Pulmonary Infection Caused by Pseudomonas aeruginosa

This study investigated the ability of a protein F vaccine to reduce macroscopic evidence of lung damage and preserve pulmonary function in immunized animals in a rat model of chronic pulmonary infection caused by Pseudomonas aeruginosa. Other membrane protein F of P aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of the PAO1 immunotype 7 strain. Rats were immunized intramuscularly with either 25 micrograms of the purified protein F or bovine serum albumin on days 0 and 14 and then challenged on day 28 via intratracheal inoculation of agar beads containing cells of an immunotype 3 clinical isolate of P aeruginosa. Also, included was a noninfected control group which received only sterile agar beads. On day 35, the lungs were excised, pulmonary compliance measured, and the lungs examined macroscopically for the presence and severity of lesions. The protein F-immunized rats had a significant (p < 0.01) reduction in the number of severe pulmonary lesions as compared with bovine serum albumin-immunized rats. Lung compliance (CL) was significantly (p < 0.001) reduced in rats which were immunized with bovine serum albumin (n = 17, CL = 0.12 +/- 0.008), whereas CL of protein F-immunized rats (n = 12, CL = 0.17 +/- 0.006) was similar to that of noninfected control rats (n = 5, CL = 0.15 +/- 0.008). This study demonstrated that a protein F vaccine has the ability to decrease macroscopic lung lesions from infection and preserve pulmonary function in actively immunized rats upon subsequent challenge with P aeruginosa in this model of chronic lung infection.

Exogenous rh-urokinase modifies inflammation and Pseudomonas aeruginosa infection in a rat chronic pulmonary infection model

The effect of recombinant human urokinase (rh-UK) in a rat model of chronic Pseudomonas aeruginosa pulmonary infection was studied. Efficacy was assessed by lung histology and quantitative bacteriology. Male Sprague-Dawley rats received 1 x 10(4) or 1 x 10(5) P. aeruginosa encapsulated in agar beads via the intratracheal route on day 1. Intratracheal administration of up to 12,500 units of rh-UK on day 21 led to a dose-dependent disappearance of viable organisms from the lungs by day 24 in rats receiving 10(4) organisms. In slightly longer term infections (30 days), rh-UK was still effective in facilitating the disappearance of the organisms from the lungs of most of the treated animals. rh-UK was effective in eliminating organisms when animals were infected with 10(4), but not 10(5) bacteria. In vitro analysis revealed that rh-UK was not directly toxic for the organisms. Histologically, lungs from short-term infected control animals exhibited acute inflammation, inflammatory cell infiltrates, and fibrin deposition. Histology of lungs from UK-treated, short-term infected rats revealed decreased airway inflammation and cellular infiltration compared with infected controls. Lungs from infected animals treated with 12,500 units of rh-UK were histologically indistinguishable from the lungs of uninfected control animals, except for the foreign body reaction. These results indicate that exogenous rh-UK may be efficacious in the treatment of pulmonary inflammation accompanying exposure to Gram-negative bacteria such as P. aeruginosa.

Loss of hypoxic pulmonary vasoconstriction in chronic pneumonia is not mediated by nitric oxide

In pulmonary inflammatory processes such as pneumonia there is diminished hypoxic pulmonary vasoconstriction (HPV). We investigated whether the attenuated HPV in pneumonia is a due to excess nitric oxide (NO) release. Sprague-Dawley rats were anesthetized, and a slurry (0.06 ml) of infected agar beads (containing 6 x 10(5) Pseudomonas aeruginosa organisms) or control (sterile) beads was then injected into a distal bronchus through a tracheotomy. After the establishment of a chronic P. aeruginosa pneumonia (7-10 days later) animals were instrumented for hemodynamic monitoring, and the response to exposure to hypoxic gas (fraction of inspired O2 = 0.08) was recorded before and after the administration of NG-monomethyl-L-arginine (L-NMMA; 50 mg/kg), an inhibitor of NO synthesis. The hypoxic pressor response, as assessed by the absolute increase in pulmonary arterial pressure (PAP) and total pulmonary resistance (TPR), was reduced in infected animals compared with control animals. The change in PAP and TPR was 8.5 +/- 0.7 and 0.053 +/- 0.007, respectively, in control animals compared with 5.9 +/- 0.5 and 0.041 +/- 0.011 in infected animals. After L-NMMA the increase in PAP and TPR during hypoxia was greater in both control and infected animals. However, treatment with L-NMMA did not affect the difference between control and infected animals. We conclude that excess release of NO does not account for the attenuated hypoxic pressor response in pneumonia.