Harry E. Gilleland

Neutrophil-mediated nitrosamine formation: Role of nitric oxide in rats

It is well known that chronic inflammation of the colon and rectum is associated with an increased risk of colorectal cancer, but the mechanisms by which inflammation promotes neoplasia remain undefined. The authors propose that inflammatory neutrophils may produce carcinogenic nitrosamines via the L-arginine-dependent formation of nitrogen oxides such as nitric oxide. Therefore, the objectives of the study were to characterize the L-arginine-dependent formation of nitrogen oxides by inflammatory (elicited) neutrophils using conditions that more closely mimic the extravascular (i.e., interstitial) compartment of the gut and to characterize the neutrophil-dependent N-nitrosation of a model amine to yield its nitrosamine derivative. In the absence of any metabolic activation, adherent, inflammatory neutrophils (2 x 10(6) cells) produced 12.8 +/- 1.4 mumol/L of nitrite during a 4-hour incubation period. Omission of L-arginine and/or inhibition of nitric oxide synthase by the addition of 1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME) resulted in 35%-78% inhibition of nitrite production, suggesting that nitrite was derived from nitric oxide. By comparison, neither circulating rat neutrophils nor elicited rat macrophages produced significant amounts of nitrite under the same conditions. Furthermore, elicited neutrophils (2 x 10(6) cells) were capable of N-nitrosating 2,3-diaminonaphthalene to yield its nitrosamine derivative 1-naphtho-2,3-triazole (282 +/- 12 nmol/L) in a time- and cell-dependent pattern similar to that of nitrite production. Addition of a variety of antioxidants (e.g., ascorbic acid, reduced glutathione, alpha-tocopherol analog), 5-aminosalicylic acid, or L-NAME resulted in 80%-85% inhibition of neutrophil-mediated nitrosamine formation. Taken together, these data suggest that inflammatory neutrophils may represent an important metabolic source of endogenous carcinogens during times of active intestinal inflammation.

Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa.

A chimeric tobacco mosaic virus (TMV) was constructed by inserting sequences representing peptide 9-14mer (TDAYNQKLSERRAN) of outer membrane (OM) protein F of Pseudomonas aeruginosa between amino acids Ser154 and Gly155 of the TMV coat protein (CP). This is the first example of TMV being used to construct a chimera containing a bacterial epitope. Mice immunized with TMV-9-14 produced anti-peptide-9-14mer-specific antibodies that reacted in whole-cell ELISA with all seven Fisher-Devlin (FD) immunotype strains of P. aeruginosa, reacted specifically by Western blotting with OM protein F extracted from all seven FD immunotypes, and were opsonic in opsonophagocytic assays. The chimeric TMV-9-14 vaccine afforded immunoprotection against challenge with wild-type P. aeruginosa in a mouse model of chronic pulmonary infection. TMV-9-14 is an excellent candidate for further development as a vaccine for possible use in humans to protect against P. aeruginosa infections.

Adaptive resistance to aminoglycoside antibiotics in Pseudomonas aeruginosa

Aminoglycoside-resistant variants of Pseudomonas aeruginosa strain PAO1 were readily selected by culturing the organism in medium containing increasing concentrations of gentamicin, tobramycin or amikacin until the strains were growing in a concentration of drug 128-fold greater than the minimal inhibitory concentration for the sensitive parent strain. These resistant strains exhibited characteristics previously associated with the impermeability type of resistance mechanism, i.e., they grew more slowly than the parent strain, the resistance was unstable in the absence of the antibiotic, and adaptation to one of the antibiotics conferred cross-resistance to other aminoglycosides. The adapted strains grew, with minimal morphological alterations, in concentrations of the various aminoglycosides that normally produced cell envelope damage, misshapen and filamentous cell formation, and cell lysis in the sensitive strain. Neither protein H1 nor phospholipid alterations appear to play a significant role in adaptive resistance to aminoglycoside antibiotics in this model system. The acquisition of adaptive resistance to the aminoglycoside antibiotics did not confer resistance to polymyxin B, another cationic antibiotic which is thought to share binding sites within the outer membrane with the aminoglycosides.

Protection against Pseudomonas aeruginosa chronic lung infection in mice by genetic immunization against outer membrane protein F (OprF) of P. aeruginosa.

ABSTRACT The Pseudomonas aeruginosa major constitutive outer membrane porin protein OprF, which has previously been shown to be a protective antigen, was targeted as a DNA vaccine candidate. TheoprF gene was cloned into plasmid vector pVR1020, and the plasmid vaccines were delivered to mice by biolistic (gene gun) intradermal inoculation. Antibody titers in antisera from immunized mice were determined by enzyme-linked immunosorbent assay, and the elicited antibodies were shown to be specifically reactive to OprF by immunoblotting. The immunoglobulin G (IgG) immune response was predominantly of the IgG1 isotype. Sera from DNA vaccine-immunized mice had significantly greater opsonic activity in opsonophagocytic assays than did sera from control mice. Following the initial immunization and two consecutive boosts, each at 2-week intervals, protection was demonstrated in a mouse model of chronic pulmonary infection byP. aeruginosa. Eight days postchallenge, both lungs were removed and examined. A significant reduction in the presence of severe macroscopic lesions, as well as in the number of bacteria present in the lungs, was seen. Based on these findings, genetic immunization withoprF has potential for development as a vaccine to protect humans against infection by P. aeruginosa.

A chimaeric plant virus vaccine protects mice against a bacterial infection.

The plant virus cowpea mosaic virus (CPMV) is an efficient carrier of foreign peptides for the generation of strong humoral immune responses. Peptides derived from both viruses and bacteria are strongly immunogenic when displayed on the surface of CPMV and elicit high titres of peptide-specific antibody. However, the protective effects of antibodies generated using bacterial epitopes in this system have yet to be demonstrated. In this study the ability of chimaeric virus particles (CVPs) to afford protection against bacterial infection was assessed. Immunization of outbred mice with CPMV expressing a peptide derived from outer-membrane protein F of Pseudomonas aeruginosa (CPMV-PAE5) generated high titres of P. aeruginosa-specific IgG that opsonized the bacteria for phagocytosis by human neutrophils and afforded protection upon challenge with two different immunotypes of P. aeruginosa in a model of chronic pulmonary infection. When examined 8 d after challenge, CVP-immunized mice had fewer severe lung lesions and fewer bacteria in their lungs compared to mice immunized with wild-type virus. Different levels of protection were seen with CPMV-PAE5 when Freunds or alum adjuvants were used. These studies highlight the ability of CVPs to generate protective immunity against infectious disease agents.

Ability of synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model

Three synthetic peptides (Nos 9, 10 and 18) representing surface-exposed, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa were each conjugated to the carriers keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), with the conjugates being used to immunize mice intranasally. Mice were also immunized intranasally with a KLH/BSA carrier control or with a peptide No. 8 conjugate as a negative control. An immunoglobulin G response reactive with P. aeruginosa whole cells was demonstrated by enzyme-linked immunosorbent assay (ELISA) of sera from mice immunized with peptide 9, 10 or 18, whereas no whole-cell reactivity by ELISA was detected in sera from mice immunized with peptide 8. Upon pulmonary challenge of immunized mice with a Fisher-Devlin immunotype 4 strain of P. aeruginosa, only those mice immunized with peptide 9 or peptide 10 had a significantly greater survival rate compared to control mice immunized with the carriers alone. Peptides 9 (TDAYNQKLSERRAN) and 10 (NATAEGRAINRRVE) have potential for further development as a protective vaccine against P. aeruginosa infections.

Vaccine efficacies of elastase, exotoxin A, and outer-membrane protein F in preventing chronic pulmonary infection by Pseudomonas aeruginosa in a rat model

The protective efficacies of eight vaccine preparations consisting of Pseudomonas aeruginosa outer-membrane protein F, elastase and exotoxin A toxoid, administered either individually or in various combinations, were determined in a rat model of chronic pulmonary infection. Rats were immunised intramuscularly at 2-week intervals (days 0, 14 and 28). On day 42, blood was collected and antisera were obtained from each vaccine group for use in an enzyme-linked immunosorbent assay which determined the titre of IgG antibodies elicited by each vaccine. Also on day 42, rats were challenged by intratracheal inoculation of a clinical isolate of P. aeruginosa encased within agar beads. On day 49, the animals were killed and the lungs were examined macroscopically for the presence of lesions and fixed for histological examination. When compared with control rats immunised with bovine serum albumin, rats immunised with protein F alone as a vaccine received significant protection against the development of severe pulmonary lesions. Elastase used alone as a vaccine provided some protection against severe lung lesions and reduced the incidence of microscopic peribronchial inflammation. However, the combination of protein F plus elastase as a vaccine did not afford protection from severe lesions, and there was an increased incidence of necrotising granulomas in the lungs from this vaccine group. Protection against lung lesions from the three-component vaccine consisting of protein F, elastase and exotoxin A toxoid was similar to that provided by the protein F vaccine. Neither macroscopic nor histological evidence showed any enhancement of protective efficacy for the three-component vaccine over that of the protein F vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant outer membrane protein F ofPseudomonas aeruginosa elicits antibodies that mediate opsonophagocytic killing, but not complement-mediated bacteriolysis, of various strains ofP. aeruginosa

Recombinant outer membrane protein F ofPseudomonas aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Rats were immunized intramuscularly with 25 μg of recombinant protein F adsorbed to aluminum hydroxide adjuvant on days 1, 14, and 28 and then challenged on day 42 via intratracheal inoculation of agar beads containing cells of a clinical isolate ofP. aeruginosa. On day 49 the lungs were examined macroscopically for the presence and severity of lesions and submitted for quantitation of the bacteria present. The recombinant protein F vaccine afforded significant protection against subsequent challenge withP. aeruginosa in the immunized rats, as compared with control rats immunized with bovine serum albumin. Antisera from the recombinant protein F-immunized rats mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells ofP. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant ofP. aeruginosa. The antisera to recombinant protein F did not promote complement-mediated bacteriolysis ofP. aeruginosa. These data demonstrate that recombinantP. aeruginosa protein F has efficacy as a protective vaccine in a rat model of chronic pulmonary infection.

DNA vaccines against chronic lung infections by Pseudomonas aeruginosa

Vaccines containing outer membrane protein F (OprF) of Pseudomonas aeruginosa are effective in reducing lesion severity in a mouse pulmonary chronic infection model. One OprF-based vaccine, called F/I, contains carboxy oprF sequences fused to oprI in an expression vector. When delivered three times biolistically by gene gun, the F/I vaccine induces protection that is antibody-mediated in outbred mice. To demonstrate the role of F/I-induced antibody-mediated immunity, B-cell-deficient [B(-)] and B-cell-intact [B(+)] mice were immunized with F/I, challenged with Pseudomonas, and examined for lesion severity. As expected, F/I-immunized B(+) mice had fewer and less severe lesions than vector-immunized B(+) mice. However, surprisingly, F/I- and vector-immunized B(-) mice were equally protected to levels similar to F/I-immunized B(+) mice. Examination of immune cell populations and cytokine levels indicated a relative increase in the quantity of CD3+ T-lymphocytes in vector- or F/I-immunized and challenged B(-) mice compared to B(+) mice. These data indicate the protective role played by cell-mediated immunity in B(-) mice, which supports our hypothesis that cell-mediated immunity can play an important role in protection against P. aeruginosa.

Low-Frequency Electromagnetic Fields Alter the Replication Cycle of MS2 Bacteriophage

Abstract. The effect of exposure to 60-Hz electromagnetic fields (EMFs) on RNA coliphage MS2 replication was studied. EMF exposure commenced when the bacterial cultures were inoculated with the phage (t = 0). In 12 experiments in which the strength of the field was 5 G, a significant delay in phage yield was found in the EMF-exposed cultures 45–65 min after inoculation, compared with control cultures. However, the EMF did not alter the final phage concentration. Experiments at 25 G (N = 5) suggested that the stronger field resulted in both impeded phage replication and increased phage yield. No differences between test groups were found in experiments involving sham-EMF exposure, thereby indicating that the results obtained with the EMFs were not due to systematic error. It appears that MS2, which codes for only four proteins, is the simplest biological system in which an EMF-induced effect has been demonstrated. The MS2 system is, therefore, conducive to follow-up studies aimed at understanding the level and nature of the underlying interaction process, and perhaps to biophysical modeling of the interaction process.