THE JOURNAL OF INFECTIOUS DISEASES. VOL. 140, NO.1. JULY 1979 © 1979 by The University of Chicago. 0022-1899/79/4001-0009$00.81
Lipopolysaccharide Pseudomonas Vaccine: Efficacy Against Pulmonary Infection with Pseudomonas aeruginosa From the Infectious Diseases Division, Department of Medicine, Peter Bent Brigham Hospital Division of the Affiliated Hospitals, Inc., Boston, Massachusetts
James E. Pennington
Pulmonary tissue is particularly vulnerable to infection with Pseudomonas aeruginosa; mortality due to pseudomonas pneumonia often exceeds 70%-80% [1-3]. Certain populations of patients, including cancer patients [1, 3, 4], cystic fibrosis patients [1, 3, 5], and patients admitted to respiratory intensive care units [2, 6-9], appear to be at high risk for pseudomonas pneumonia. The unsatisfactory prognosis for such patients, even with currently available antibiotics, plus the predictable incidence of pseudomonas pneumonia in specific groups of patients, has led to interest among physicians in immunoprophylaxis or immunotherapy for pseudomonas pneumonia [3, 7, 10]. The concept of an immunologic approach to the problem of pseudomonas infections is not
new [11], but to date the majority of studies utilizing pseudomonas vaccines have dealt primarily with burn patients [12] rather than specifically with the groups at highest risk for pulmonary infections. While studies in rabbits have demonstrated that pseudomonas lipopolysaccharide (LPS) antigens can elicit a specific antibody response in respiratory secretions [13, 14] and other studies in neutropenic dogs have shown that experimentally induced pseudomonas pneumonia is associated with higher survival rates when antiserum to Pseudomonas is given [15], to date only one clinical study has suggested that LPS pseudomonas vaccine may offer specific protection for respiratory tissue [7]. The lung is an immunologically unique organ, with particular local host defense mechanisms operating, at times, independent of systemic responses [16, 17]. Thus, the potential value of any pseudomonas vaccine for protection of respiratory tissues against pseudomonas pneumonia must be specifically evaluated for local pulmonary infections. Until now, the lack of a reproducible model of fatal pseudomonas pneumonia in normal (immunologically intact) animals has prevented a thorough in vivo laboratory evaluation of pseudomonas vaccines for respiratory protection against Pseudomonas. In this study, a particularly virulent strain of P. aeruginosa was
Received for publication May 3, 1978, and in revised form January 31, 1979. This study was supported by a grant from the Cystic Fibrosis Foundation. The author thanks Diana Kuchmy for technical assistance and Dr. William F. Hickey and Betty Flammio, Department of Pathology, Peter Bent Brigham Hospital, for assistance with the histopathologic studies. He also thanks Drs. Mike Fisher and Carl Heifetz, Parke, Davis and Co., Detroit, Michigan, for their contributions to this project and Viki Fowler for help in preparation of the manuscript. Please address requests for reprints to Dr. James E. Pennington, Department of Medicine, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115.
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Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on September 4, 2015
Pneumonia due to Pseudomonas aeruginosa occurs with increased frequency and high mortality in certain populations of patients. The potential of vaccination with a heptavalent lipopolysaccharide pseudomonas vaccine for specific protection of respiratory tissues from infection with Pseudomonas was evaluated with a guinea pig model of experimental pseudomonas pneumonia. Animals routinely responded to vaccination with a fourfold rise in titer of serum hemagglutinating antibody to Pseudomonas. Of 25 control animals, all but nine died after lung challenge with Pseudomonas, whereas vaccinated animals had a greater survival rate (22 of 25 animals survived; P < 0.01). Rates of clearance of viable Pseudomonas from lung tissue were significantly greater in vaccinated animals than in controls during the first 6 hr after infection. Both gross and microscopic findings of lung tissue damage from pseudomonas pneumonia were less in vaccinated than in control animals. Thus, lipopolysaccharide pseudomonas vaccine appears to produce a local protective response in respiratory tissue against Pseudomonas.
74
found to produce fatal hemorrhagic pneumonia in normal guinea pigs. The ability of a heptavalent LPS pseudomonas vaccine to increase the rate of survival in animals with pneumonia, to enhance clearance of viable Pseudomonas from the lung, and to decrease lung tissue damage in survivors of pneumonia was studied in this guinea pig model of experimental pseudomonas pneumonia.
A nimals. Hartley strain guinea pigs weighing 400 g were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Animals were housed in standard animal cages and were fed routine laboratory chow (Ralston-Purina, St. Louis, 1\110.) and water. P. aeruginosa. The strain of P. aeruginosa employed in this study, strain PD05141 (designated herein as P-4), was obtained from Drs. Mike Fisher and Carl Heifetz, Parke, Davis and Co., Detroit, Mich. This strain (Fisher immunotype 4) was isolated from a patient with sepsis and was maintained on brucella agar slants at 4 C. Analysis of strain P-4 revealed that it was resistant to incubation for 1 hr in freshly obtained guinea pig whole serum and that it produced only small amounts of exotoxin A (assayed by Dr. 1\11. Pollack, Naval Medical Research Institute, Bethesda, Md.). For each experiment, four loops of strain P-4 were placed in 50 ml of trypticase soy broth and incubated overnight at 37 C. Pseudomonas organisms were then sedimented by centrifugation at 1,800 g for 5 min and resuspended in 20 ml of 0.9% saline by vortex agitation. The centrifugation and washing were repeated three times, and the final suspension was standardized to a known concentration by measurement of the OD at 650 nm with a Gilford 240 spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio). Standard serial dilution pour-plate cultures were done to verify the exact number of cfu/rnl. Suspensions were adjusted by dilution with saline to the concentration of Pseudomonas desired for each experiment. Vaccine and vaccination protocol. The P. aeruginosa vaccine utilized in this study was a preparation of heptavalent LPS (Pseudogen,"
lots no. 41667 and 42083; Parke-Davis) [18]. Fisher immunotype 4 antigen (corresponding to challenge strain P-4) was included in this vaccine. Pseudogen was provided in 1.2-ml vials, containing 0.85 mg of LPS pseudomonas antigen suspended in 0.01 % thimerosal. Vaccine was stored at 4 C and diluted 1: lOin 0.9% saline before use. The vaccination protocol employed in this study was a series of six im injections given over a two-week period. Each injection contained 50 /Lg of LPS pseudomonas antigen/kg of animal body weight, and the injections were given in alternating flank muscles. The animals tolerated this regimen well, with no local abscesses, illness, or unexpected deaths occurring. For each series of vaccinations, equivalent numbers of control animals received a series of six im injections of sterile 0.9% saline. Immune response to vaccination. The ability of Pseudogen to elicit a humoral antibody response in guinea pig serum and bronchial secretions was determined. In addition, titers of immunotype 4 antibody were correlated with protection of guinea pigs from pneumonia due to P. aeruginosa type 4. Serum was separated from blood obtained from vaccinated and control animals via cardiac puncture during the week following' vaccination, and the specimens were stored at -20 C until assayed. In addition, six vaccinated and six control animals were killed (by pentobarbital given ip) one week after completion of the vaccine protocol, and bronchial lavage was performed with use of a previously described technique [19]. Samples of bronchial lavage (-80 ml per animal) were concentrated to 5-ml volumes at 4 C using a positivepressure ultrafiltration device (Amicon Corp., Lexington, Mass.) with a U1\II-10 filter. The OD values (at 280 nm) of the concentrated lavages (dilution, I: 10) were compared and were similar (mean OD ± SE, 0.27 ± 0.04). Lavage specimens were frozen at -20 C until assayed. Antibody response was determined using a previously described passive HA assay [20] (performed by Dr. Carl Heifetz). The HA antibody titer was read as the reciprocal of the highest dilution of serum showing I + red blood cell agglutination. HAantibody titers were determined for all specimens both with and without reduction with 2-mercaptoethanol (2-ME) [21].
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on September 4, 2015
Materials and Methods
Pennington
LPS Pseudomonas Vaccine
The lungs of 25 control and 25 vaccinated guinea pigs were challenged with 1 X 108 cfu of strain P-4/o.5 ml of saline five to seven days after completion of the vaccination protocol. The animals received no further therapy and were left in their cages with food and water and observed for survival. Clearance of Pseudomonas from lungs. The capacity of vaccination with Pseudomonas to influence pulmonary clearance rates for viable Pseudomonas was determined. For these studies, guinea pig lungs were challenged with a sublethal dose of Pseudomonas (5 X 106 cfu/O.5 ml) to ensure survival over the monitoring period. Groups of vaccinated and control animals were challenged at zero-time and were then killed with pentobarbital given ip 3, 6, or 24 hr after infection. In addition, selected animals were killed immediately after infection so that base-line lung cultures could be obtained. Immediately after sacrifice, the neck and thorax were opened, and the trachea, lungs, and heart removed in toto using aseptic technique. The right lung was then resected and placed in a commercial grade Waring blender (Waring Products, New Hartford, Conn.) containing 25 ml of sterile saline. Lungs were homogenized at low speed for 1 min, and quantitative cultures of homogenate were made using serial dilutions of homogenate placed in pour plates with trypticase soy agar. Colony growth at 24 hr was easily read on a Quebec Colony Counter (American Optical, Buffalo, N.Y.), and the number of cfu rrnl of lung homogenate was determined. Gross and histopathologic observations. In addition to the quantitative lung cultures obtained 3, 6, and 24 hr after sublethal challenge with Pseudomonas, gross pathologic changes were also recorded at these times. Furthermore, the remaining (i.e., left) lungs from animals killed at 24 hr were processed for microscopic examination of tissue. After the lungs of these animals were removed, the right mainstem bronchus was tied off and the right lung was resected distal to the ligature. This lung was used for quantitative cultures (see above). The trachea was then cannulated with a no. 18 Ranfac special needle (Randell-Faichney, Boston, Mass.), and the left lung was infused with --5 ml of 1070 formalin. The lung was carefully expanded to avoid over-
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on September 4, 2015
Experimental pseudomonas pneumonia. A method for inducing pseudomonas pneumonia in guinea pigs with use of intratracheal instillations of suspensions of Pseudomonas has recently been described [22]. In brief, 400-g Hartley strain guinea pigs were anesthetized with pentobarbital given ip, and their tracheas were surgically exposed. P. aeruginosa, suspended in 0.9% saline, was instilled intratracheally via a syringe with a no. 25 needle. After the trachea was briefly occluded proximally to prevent coughing, the neck was sutured closed. The animals were then positioned upright until awake to allow distal distribution of the bacteria. A bilateral hemorrhagic pneumonia was reliably induced with this technique [22]. Instillations of sterile saline did not result in pulmonary pathology in control studies [22]. The rationale for utilizing direct instillations rather than aerosols has also been explained previously [19, 22]. Studies of survival. It was initially necessary to establish that an experimental pneumonia induced by P. aeruginosa strain P-4 could result in death in normal (immunologically intact) guinea pigs, and if so, what challenge dose was needed. Four separate groups of four normal guinea pigs underwent lung challenge with a different concentration of strain P-4. The challenge doses selected were 1 X 107 cfu, 5 X 107 cfu, 1 X 108 du, and 5 X 108 du (in a volume of 0.5 ml), The number of deaths in each group was zero, one, four, and four, respectively. Autopsies revealed extensive bilateral hemorrhagic pneumonia in all fatal cases, and cultures of lung tissue resulted in a heavy growth of P. aeruginosa. Since a suspension of 1 X 108 du of strain P-4 in 0.5 ml of saline resulted in fatal outcome for the majority of normal animals, this dose was selected for the vaccine survival trials. To prove that deaths were related to pseudomonas infection, rather than simply a reaction of lung tissues to, the instillation of toxic pseudomonas antigen, aliquots of Pseudomonas containing 1 X 108 du in a volume of 0.5 ml were heat-killed (60 C for 90 min) prior to lung challenge. These inocula of heat-killed Pseudomonas were not fatal for three normal animals. The ability of vaccination with Pseudogen to improve rates of survival among animals with pseudomonas pneumonia was determined next.
75
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Pennington
inflation, and the trachea was tied off. Lungs were fixed in 10% formalin for 48 hr, then transected longitudinally from apex to base and embedded in paraffin for sectioning. Each half of the lung was sectioned at four levels so that a total of eight different sections of lung were available for evaluation. Tissue sections were stained with hematoxylin and eosin. Results
Table 1.
HA antibody response of immunologically intact guinea pigs to vaccination with heptavalent pseudomonas lipopolysaccharide (Pseudogen.P ParkeDavis, Detroit, Mich.). Fisher immunotype, fluid tested All seven Serum Bronchial secretions Type 4 Serum Bronchial secretions
Group Control*
Vaccinatedt
Lipopolysaccharide Pseudomonas Vaccine: Efficacy Against Pulmonary Infection with Pseudomonas aeruginosa From the Infectious Diseases Division, Department of Medicine, Peter Bent Brigham Hospital Division of the Affiliated Hospitals, Inc., Boston, Massachusetts
James E. Pennington
Pulmonary tissue is particularly vulnerable to infection with Pseudomonas aeruginosa; mortality due to pseudomonas pneumonia often exceeds 70%-80% [1-3]. Certain populations of patients, including cancer patients [1, 3, 4], cystic fibrosis patients [1, 3, 5], and patients admitted to respiratory intensive care units [2, 6-9], appear to be at high risk for pseudomonas pneumonia. The unsatisfactory prognosis for such patients, even with currently available antibiotics, plus the predictable incidence of pseudomonas pneumonia in specific groups of patients, has led to interest among physicians in immunoprophylaxis or immunotherapy for pseudomonas pneumonia [3, 7, 10]. The concept of an immunologic approach to the problem of pseudomonas infections is not
new [11], but to date the majority of studies utilizing pseudomonas vaccines have dealt primarily with burn patients [12] rather than specifically with the groups at highest risk for pulmonary infections. While studies in rabbits have demonstrated that pseudomonas lipopolysaccharide (LPS) antigens can elicit a specific antibody response in respiratory secretions [13, 14] and other studies in neutropenic dogs have shown that experimentally induced pseudomonas pneumonia is associated with higher survival rates when antiserum to Pseudomonas is given [15], to date only one clinical study has suggested that LPS pseudomonas vaccine may offer specific protection for respiratory tissue [7]. The lung is an immunologically unique organ, with particular local host defense mechanisms operating, at times, independent of systemic responses [16, 17]. Thus, the potential value of any pseudomonas vaccine for protection of respiratory tissues against pseudomonas pneumonia must be specifically evaluated for local pulmonary infections. Until now, the lack of a reproducible model of fatal pseudomonas pneumonia in normal (immunologically intact) animals has prevented a thorough in vivo laboratory evaluation of pseudomonas vaccines for respiratory protection against Pseudomonas. In this study, a particularly virulent strain of P. aeruginosa was
Received for publication May 3, 1978, and in revised form January 31, 1979. This study was supported by a grant from the Cystic Fibrosis Foundation. The author thanks Diana Kuchmy for technical assistance and Dr. William F. Hickey and Betty Flammio, Department of Pathology, Peter Bent Brigham Hospital, for assistance with the histopathologic studies. He also thanks Drs. Mike Fisher and Carl Heifetz, Parke, Davis and Co., Detroit, Michigan, for their contributions to this project and Viki Fowler for help in preparation of the manuscript. Please address requests for reprints to Dr. James E. Pennington, Department of Medicine, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115.
73
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on September 4, 2015
Pneumonia due to Pseudomonas aeruginosa occurs with increased frequency and high mortality in certain populations of patients. The potential of vaccination with a heptavalent lipopolysaccharide pseudomonas vaccine for specific protection of respiratory tissues from infection with Pseudomonas was evaluated with a guinea pig model of experimental pseudomonas pneumonia. Animals routinely responded to vaccination with a fourfold rise in titer of serum hemagglutinating antibody to Pseudomonas. Of 25 control animals, all but nine died after lung challenge with Pseudomonas, whereas vaccinated animals had a greater survival rate (22 of 25 animals survived; P < 0.01). Rates of clearance of viable Pseudomonas from lung tissue were significantly greater in vaccinated animals than in controls during the first 6 hr after infection. Both gross and microscopic findings of lung tissue damage from pseudomonas pneumonia were less in vaccinated than in control animals. Thus, lipopolysaccharide pseudomonas vaccine appears to produce a local protective response in respiratory tissue against Pseudomonas.
74
found to produce fatal hemorrhagic pneumonia in normal guinea pigs. The ability of a heptavalent LPS pseudomonas vaccine to increase the rate of survival in animals with pneumonia, to enhance clearance of viable Pseudomonas from the lung, and to decrease lung tissue damage in survivors of pneumonia was studied in this guinea pig model of experimental pseudomonas pneumonia.
A nimals. Hartley strain guinea pigs weighing 400 g were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Animals were housed in standard animal cages and were fed routine laboratory chow (Ralston-Purina, St. Louis, 1\110.) and water. P. aeruginosa. The strain of P. aeruginosa employed in this study, strain PD05141 (designated herein as P-4), was obtained from Drs. Mike Fisher and Carl Heifetz, Parke, Davis and Co., Detroit, Mich. This strain (Fisher immunotype 4) was isolated from a patient with sepsis and was maintained on brucella agar slants at 4 C. Analysis of strain P-4 revealed that it was resistant to incubation for 1 hr in freshly obtained guinea pig whole serum and that it produced only small amounts of exotoxin A (assayed by Dr. 1\11. Pollack, Naval Medical Research Institute, Bethesda, Md.). For each experiment, four loops of strain P-4 were placed in 50 ml of trypticase soy broth and incubated overnight at 37 C. Pseudomonas organisms were then sedimented by centrifugation at 1,800 g for 5 min and resuspended in 20 ml of 0.9% saline by vortex agitation. The centrifugation and washing were repeated three times, and the final suspension was standardized to a known concentration by measurement of the OD at 650 nm with a Gilford 240 spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio). Standard serial dilution pour-plate cultures were done to verify the exact number of cfu/rnl. Suspensions were adjusted by dilution with saline to the concentration of Pseudomonas desired for each experiment. Vaccine and vaccination protocol. The P. aeruginosa vaccine utilized in this study was a preparation of heptavalent LPS (Pseudogen,"
lots no. 41667 and 42083; Parke-Davis) [18]. Fisher immunotype 4 antigen (corresponding to challenge strain P-4) was included in this vaccine. Pseudogen was provided in 1.2-ml vials, containing 0.85 mg of LPS pseudomonas antigen suspended in 0.01 % thimerosal. Vaccine was stored at 4 C and diluted 1: lOin 0.9% saline before use. The vaccination protocol employed in this study was a series of six im injections given over a two-week period. Each injection contained 50 /Lg of LPS pseudomonas antigen/kg of animal body weight, and the injections were given in alternating flank muscles. The animals tolerated this regimen well, with no local abscesses, illness, or unexpected deaths occurring. For each series of vaccinations, equivalent numbers of control animals received a series of six im injections of sterile 0.9% saline. Immune response to vaccination. The ability of Pseudogen to elicit a humoral antibody response in guinea pig serum and bronchial secretions was determined. In addition, titers of immunotype 4 antibody were correlated with protection of guinea pigs from pneumonia due to P. aeruginosa type 4. Serum was separated from blood obtained from vaccinated and control animals via cardiac puncture during the week following' vaccination, and the specimens were stored at -20 C until assayed. In addition, six vaccinated and six control animals were killed (by pentobarbital given ip) one week after completion of the vaccine protocol, and bronchial lavage was performed with use of a previously described technique [19]. Samples of bronchial lavage (-80 ml per animal) were concentrated to 5-ml volumes at 4 C using a positivepressure ultrafiltration device (Amicon Corp., Lexington, Mass.) with a U1\II-10 filter. The OD values (at 280 nm) of the concentrated lavages (dilution, I: 10) were compared and were similar (mean OD ± SE, 0.27 ± 0.04). Lavage specimens were frozen at -20 C until assayed. Antibody response was determined using a previously described passive HA assay [20] (performed by Dr. Carl Heifetz). The HA antibody titer was read as the reciprocal of the highest dilution of serum showing I + red blood cell agglutination. HAantibody titers were determined for all specimens both with and without reduction with 2-mercaptoethanol (2-ME) [21].
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on September 4, 2015
Materials and Methods
Pennington
LPS Pseudomonas Vaccine
The lungs of 25 control and 25 vaccinated guinea pigs were challenged with 1 X 108 cfu of strain P-4/o.5 ml of saline five to seven days after completion of the vaccination protocol. The animals received no further therapy and were left in their cages with food and water and observed for survival. Clearance of Pseudomonas from lungs. The capacity of vaccination with Pseudomonas to influence pulmonary clearance rates for viable Pseudomonas was determined. For these studies, guinea pig lungs were challenged with a sublethal dose of Pseudomonas (5 X 106 cfu/O.5 ml) to ensure survival over the monitoring period. Groups of vaccinated and control animals were challenged at zero-time and were then killed with pentobarbital given ip 3, 6, or 24 hr after infection. In addition, selected animals were killed immediately after infection so that base-line lung cultures could be obtained. Immediately after sacrifice, the neck and thorax were opened, and the trachea, lungs, and heart removed in toto using aseptic technique. The right lung was then resected and placed in a commercial grade Waring blender (Waring Products, New Hartford, Conn.) containing 25 ml of sterile saline. Lungs were homogenized at low speed for 1 min, and quantitative cultures of homogenate were made using serial dilutions of homogenate placed in pour plates with trypticase soy agar. Colony growth at 24 hr was easily read on a Quebec Colony Counter (American Optical, Buffalo, N.Y.), and the number of cfu rrnl of lung homogenate was determined. Gross and histopathologic observations. In addition to the quantitative lung cultures obtained 3, 6, and 24 hr after sublethal challenge with Pseudomonas, gross pathologic changes were also recorded at these times. Furthermore, the remaining (i.e., left) lungs from animals killed at 24 hr were processed for microscopic examination of tissue. After the lungs of these animals were removed, the right mainstem bronchus was tied off and the right lung was resected distal to the ligature. This lung was used for quantitative cultures (see above). The trachea was then cannulated with a no. 18 Ranfac special needle (Randell-Faichney, Boston, Mass.), and the left lung was infused with --5 ml of 1070 formalin. The lung was carefully expanded to avoid over-
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on September 4, 2015
Experimental pseudomonas pneumonia. A method for inducing pseudomonas pneumonia in guinea pigs with use of intratracheal instillations of suspensions of Pseudomonas has recently been described [22]. In brief, 400-g Hartley strain guinea pigs were anesthetized with pentobarbital given ip, and their tracheas were surgically exposed. P. aeruginosa, suspended in 0.9% saline, was instilled intratracheally via a syringe with a no. 25 needle. After the trachea was briefly occluded proximally to prevent coughing, the neck was sutured closed. The animals were then positioned upright until awake to allow distal distribution of the bacteria. A bilateral hemorrhagic pneumonia was reliably induced with this technique [22]. Instillations of sterile saline did not result in pulmonary pathology in control studies [22]. The rationale for utilizing direct instillations rather than aerosols has also been explained previously [19, 22]. Studies of survival. It was initially necessary to establish that an experimental pneumonia induced by P. aeruginosa strain P-4 could result in death in normal (immunologically intact) guinea pigs, and if so, what challenge dose was needed. Four separate groups of four normal guinea pigs underwent lung challenge with a different concentration of strain P-4. The challenge doses selected were 1 X 107 cfu, 5 X 107 cfu, 1 X 108 du, and 5 X 108 du (in a volume of 0.5 ml), The number of deaths in each group was zero, one, four, and four, respectively. Autopsies revealed extensive bilateral hemorrhagic pneumonia in all fatal cases, and cultures of lung tissue resulted in a heavy growth of P. aeruginosa. Since a suspension of 1 X 108 du of strain P-4 in 0.5 ml of saline resulted in fatal outcome for the majority of normal animals, this dose was selected for the vaccine survival trials. To prove that deaths were related to pseudomonas infection, rather than simply a reaction of lung tissues to, the instillation of toxic pseudomonas antigen, aliquots of Pseudomonas containing 1 X 108 du in a volume of 0.5 ml were heat-killed (60 C for 90 min) prior to lung challenge. These inocula of heat-killed Pseudomonas were not fatal for three normal animals. The ability of vaccination with Pseudogen to improve rates of survival among animals with pseudomonas pneumonia was determined next.
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Pennington
inflation, and the trachea was tied off. Lungs were fixed in 10% formalin for 48 hr, then transected longitudinally from apex to base and embedded in paraffin for sectioning. Each half of the lung was sectioned at four levels so that a total of eight different sections of lung were available for evaluation. Tissue sections were stained with hematoxylin and eosin. Results
Table 1.
HA antibody response of immunologically intact guinea pigs to vaccination with heptavalent pseudomonas lipopolysaccharide (Pseudogen.P ParkeDavis, Detroit, Mich.). Fisher immunotype, fluid tested All seven Serum Bronchial secretions Type 4 Serum Bronchial secretions
Group Control*
Vaccinatedt
