Design and evaluation of a low-cost respiratory monitoring device for use with anaesthetized animals.

This report describes a simple, non-invasive electronic device that employs a compact accelerometer integrated circuit to transduce movements in the chest wall of an anaesthetized animal into an analogue signal that can be used to calculate the rate and relative depth of respiration. The device requires amplification by signal processing hardware/software which are common to most experimental laboratories. We assessed the sensitivity of the device by its ability to detect changes in respiratory patterns produced by modulating the depth of anaesthesia in isoflurane-anaesthetized Wistar rats. It is widely accepted that many anaesthetic agents affect respiratory patterns, especially respiratory rate (RR), which is often used as an important index of anaesthetic depth. Respiratory parameters obtained with the device were compared with concurrently recorded electroencephalographic and cardiac measures. Different concentrations of anaesthetic agent produced four depths of anaesthesia, identified using established electroencephalographic criteria. The accelerometer was attached easily and securely to the location of maximal chest wall movement and produced a strong respiratory signal that was detectable in all four anaesthetic stages. Deepening the anaesthesia produced a gradual decrease in RR, a decrease in dominant spectral frequency of the electroencephalogram (EEG) but no change in the heart rate. There was a significant correlation between RR and the dominant spectral frequency of the EEG, indicating that one useful application of the monitor could be to identify anaesthetic stages. The results demonstrate that respiratory parameters can be recorded using a simply constructed, low-cost device and suggest an application in the monitoring of anaesthetic depth.

Prophylactic and therapeutic efficacy of immunoglobulin G antibodies to Pseudomonas aeruginosa lipopolysaccharide against murine experimental corneal infection.

PURPOSE: To evaluate the efficacy of lipopolysaccharide (LPS)-specific antibodies administered prophylactically or therapeutically to protect against corneal challenge with Pseudomonas aeruginosa. METHODS: The prophylactic efficacy of active immunization with purified P. aeruginosa LPS was evaluated in a murine corneal-scratch model of P. aeruginosa keratitis. The same model was used to evaluate both the prophylactic and the therapeutic efficacy of systemic passive transfer of variable region-identical, isotype-switched, LPS-specific, murine immunoglobulin M (IgM) and immunoglobulin G (IgG) monoclonal antibodies (mAbs). The mAbs were injected intraperitoneally at various times either before or after corneal challenge and the corneal response was monitored macroscopically. In addition, immune rabbit sera were used to evaluate the efficacy of treatment. RESULTS: Active immunization with homologous, but not heterologous, LPS before challenge reduced the severity of corneal disease and protected challenged mice against permanent corneal damage. Passive transfer of the LPS-specific IgM mAb 1F6 before challenge did not prevent corneal damage at any dose tested and had no effect on the course of disease. However, results of dose-response studies of the passive transfer of a variable region-identical IgG2b mAb, 2H3, before challenge indicated a 50% protective dose of 11.8 micrograms. When mAb 2H3 was administered at a dose of 50 micrograms before challenge and the challenge inoculum was increased, all mice were protected from corneal damage up to a challenge inoculum of 2.2 x 10(8) CFU/eye. When given 2 or 4 hours after corneal challenge with P. aeruginosa strain 6294 (which invades corneal epithelial cells during infection) but not when given at 8 or 24 hours, 50 micrograms of mAb 2H3 conferred significant protection (P < 0.05). The maximal interval after challenge during which this antibody could be administered and still protect 50% of mice was calculated by probit analysis to be 9.4 hours. Administration of homologous LPS-specific rabbit antiserum to mice at various times after challenge with P. aeruginosa strain 6206 (which is cytotoxic to corneal epithelial cells and does not remain in these cells during infection) resulted in significant protection when administered 4 or 8 hours after infection. Although probit analysis could not be performed with the available data, 50% of mice were completely protected when the antiserum was given up to 24 hours after challenge. CONCLUSIONS: In an experimental model of P. aeruginosa keratitis, systematically delivered IgG antibodies directed against the O-side-chain antigens of P. aeruginosa, LPS conferred protection against severe corneal damage when administered both prophylactically and therapeutically.

Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa.

PURPOSE: Pseudomonas aeruginosa has been observed to be adherent to and inside epithelial cells during experimental corneal infection. The authors identified bacterial ligands involved in adherence and entry of P. aeruginosa into corneal epithelial cells. METHODS: In vitro gentamicin survival assays were used to determine the intracellular survival of a panel of P. aeruginosa mutants. Strains (10(6) to 10(7) colony-forming units) were added to primary cultures of rabbit corneal epithelial cells (approximately 10(5)/well) for 3 hours, nonadherent bacteria were washed away, and extracellular bacteria were killed with gentamicin. The antibiotic was then washed away, and epithelial cells were lysed with 0.5% Triton X-100 to release internalized bacteria. Bacterial association (sum of bound and internalized bacteria) was measured by the omission of gentamicin. Similar assays were carried out with whole mouse eyes in situ. RESULTS: A lipopolysaccharide core with an exposed terminal glucose residue was found to be necessary for maximal association and entry of P. aeruginosa into corneal cells. Bacterial pili and flagella were not involved. Mutants of P. aeruginosa strains that do not produce an LPS core with a terminal glucose residue had a significantly lower level of association with (approximately 50%) and ingestion by ( > 90%, P < 0.01) corneal cells than did strains with this characteristic. Complementation of the LPS productions defect by plasmid-borne DNA returned association and ingestion to near parental levels. Lipopolysaccharides and delipidated oligosaccharides with a terminal glucose residue in the core inhibited bacterial association and entry into corneal cells. Experiments using P. aeruginosa LPS mutants and corneal cells on whole mouse eyes confirmed the role of the LPS core in cellular entry. CONCLUSIONS: Corneal epithelial cells bind and internalized P. aeruginosa by the exposed LPS core.

Oviductal, not oviducal.

Both etymologically and in literal meaning the term "oviductal" is overwhelmingly preferable to "oviducal."

Inhibition of bacterial adherence to host tissue does not markedly affect disease in the murine model of Pseudomonas aeruginosa corneal infection.

The prevention of bacterial infections by the inhibition of binding to host tissues is an oft-touted approach, but few studies with appropriate models of infection have tested its feasibility. Pseudomonas aeruginosa causes severe corneal infections in mice after inoculations with low doses, and infection is thought to depend upon an initial adherence of the bacteria to corneal cells. In vitro, adherence to corneal cells is mediated to a large degree by the complete-outer-core oligosaccharide of the bacterial lipopolysaccharide (LPS). However, bacteria adhering to tissues in vivo are difficult to differentiate from nonadherent bacteria. Since a direct correlate of P. aeruginosa adherence to corneal epithelial cells is the degree to which these cells internalize P. aeruginosa, the level of adherence in vivo can be approximated by measuring P. aeruginosa ingestion by cells by using gentamicin exclusion assays. To determine the degree to which inhibition of the corneal cell adherence affects the course of infection and disease in the murine model, we evaluated the ability of LPS-outer-core oligosaccharide to inhibit bacterial association and entry into corneal cells and to modulate the development of disease. Mice were anesthetized, and their corneas were scratched and inoculated with virulent P. aeruginosa 6294 or PAO1, along with either 50 microg of oligosaccharide derived from LPS from P. aeruginosa PAC557 (complete outer core but no O side chains) or oligosaccharide derived from LPS of P. aeruginosa PAC1RalgC::tet (incomplete-core oligosaccharide). After 4 h, there were no differences between groups in the counts of infecting and internalized bacteria. At 24 h, the complete-core oligosaccharide decreased the levels of bacteria per eye by 70 to 99.7% compared with the levels achieved by including the incomplete-core oligosaccharide in the infectious inoculum. Epithelial cell ingestion of bacteria was comparably affected. However, the effect on disease was modest and only evident at lower challenge doses that elicited mild disease in controls and when the bacterial association and ingestion were inhibited by >99%. Overall, it appears that in the murine model of P. aeruginosa corneal infection at challenge doses of bacteria 10-fold or greater than the minimal amount needed to cause disease, the absolute level of inhibition of bacterial adherence is insufficient to reduce the bacterial counts below that which elicits disease.

Kinetics of serum and ocular antibody responses in susceptible mice that received a secondary corneal infection with Pseudomonas aeruginosa.

When susceptible C57BL/6J mice were infected with Pseudomonas aeruginosa in one eye and then reinfected in the previously uninfected contralateral control eye either 4 or 8 weeks after the primary infection, approximately 20 to 30% of the mice receiving a 4-week reinfection regimen restored corneal clarity within 4 weeks, while almost all of the 8-week-reinfected mice restored corneal clarity within 3 to 6 days postinfection. However, the rate of bacterial clearance was the same in both sets of mice despite the presence of opsonophagocytic antibodies only in the 8-week-reinfected mice. As determined by enzyme-linked immunosorbent assay, immunoglobulin G was the major immunoglobulin in both serum and ocular tissue of both mouse sets, and the immunoglobulin G level was two- to fourfold higher after the 8-week secondary infection than in the 4-week-reinfected mice.

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection.

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.

Functional properties of isotype-switched immunoglobulin M (IgM) and IgG monoclonal antibodies to Pseudomonas aeruginosa lipopolysaccharide.

Controversy exists regarding isotype-related differences in the antibacterial and protective properties of lipopolysaccharide (LPS)-specific antibodies of the immunoglobulin M (IgM) class and various IgG subclasses. To clarify this issue, a murine hybridoma secreting IgM monoclonal antibody (MAb) specific for the O polysaccharide of Pseudomonas aeruginosa serogroup O6 LPS was class switched, by sib selection, to produce an IgG3 MAb with identical specificity and variable region heavy and light chain nucleotide sequences. This IgG3-secreting cell line was further switched to the production of O-specific, variable region-identical IgG1, IgG2b, and IgG2a MAbs. Functional comparisons of these LPS-specific IgM and IgG MAb isotypes revealed similar LPS binding, opsonic, and protective activities. Relatively minor isotype-related differences in levels of efficiency of MAb-mediated, complement-dependent opsonophagocytic killing (IgM > IgG2a > IgG3 > IgG2b > > IgG1) were not associated with corresponding differences in in vivo functions. These findings, in conjunction with previously published data, support a cautious approach to generic conclusions regarding the immunotherapeutic superiority of LPS-specific antibodies belonging to either the IgM or IgG class or to a particular IgG subclass.

Production and characterization of a set of mouse-human chimeric immunoglobulin G (IgG) subclass and IgA monoclonal antibodies with identical variable regions specific for Pseudomonas aeruginosa serogroup O6 lipopolysaccharide.

The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy.

Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection.

Pseudomonas aeruginosa is considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have demonstrated that some strains of P. aeruginosa are able to invade corneal cells during experimental bacterial keratitis in mice. Although intracellular bacteria were detectable 15 min after inoculation, the number of intracellular bacteria increased in a time-dependent manner over a 24-h period. Levels of invasion were similar when bacteria were grown as a biofilm on solid medium and when they were grown in suspension. Intracellular bacteria survived in vitro for at least 24 h, although only minimal bacterial multiplication within cells was observed. P. aeruginosa PAK and Escherichia coli HB101 did not cause disease in this model and were not isolated from corneas after 24 h even when an inoculum of 10(8) CFU was applied. Transmission electron microscopy of corneal epithelium from eyes infected for 8 h revealed that intracellular bacteria were present within membrane-bound vacuoles, which suggests that bacterial entry was an endocytic process. At 24 h, the observation of many bacteria free in the cytoplasm indicated that P. aeruginosa was able to escape the endocytic vacuole. The ability of some P. aeruginosa strains to invade corneal epithelial cells may contribute to the pathogenesis or to the progression of disease, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.

Kinetics of serum, tear, and corneal antibody responses in resistant and susceptible mice intracorneally infected with Pseudomonas aeruginosa.

The studies described here are aimed at determining the kinetics of antibody responses specific to Pseudomonas aeruginosa ATCC 19660 in sera, tears, and corneas of naturally resistant DBA/2 mice and susceptible C57BL/6 mice after intracorneal infection. Immunoglobulin (IgG) and IgM responses in sera were significantly greater in DBA/2 mice for the first 2 weeks postinfection. Little or no IgA was detected in the sera of mice from either strain. IgG was the predominant immunoglobulin class present in the corneas of the infected eyes from both mouse strains. However, differences in both the magnitude and the kinetics of the corneal IgG responses were noted between mouse strains. The kinetics of the corneal IgG responses were more similar to those of the serum IgG response than to those of the tear IgG response. Tear antibody responses in DBA/2 mice differed from those of C57BL/6 mice in two ways. First, there was a sharp increase in tear IgG levels 2 weeks after infection in DBA/2 mice that was not present in C57BL/6 mice. Second, IgA levels present in tears from the infected eyes of C57BL/6 mice dropped to nearly preinfection levels after the first week, whereas in DBA/2 mice, IgA levels remained elevated in the infected eyes after the first week. Determination of P. aeruginosa-specific antibody responses in the uninfected, contralateral control eyes revealed that IgA was detectable in the tears but not in the corneas of DBA/2 mice. Very little IgA was detected in the tears of the uninfected eyes of C57BL/6 mice. IgG was the only immunoglobulin class present in the uninfected corneas in both mouse strains tested. These results suggest that ocular IgA was made locally, whereas most ocular IgG may have originated from the serum, with some possible local synthesis. These immunological results indicate that DBA/2 and C57BL/6 mice respond differently to corneal challenge with P. aeruginosa.

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa.

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.

Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa.

We have reported that some strains of Pseudomonas aeruginosa can enter corneal epithelial cells during experimental murine eye infection and when the cells are cultured in vitro. Following invasion, both the host cell and the intracellular bacteria can remain viable for up to 24 h. Others have reported that toxin-mediated damage of epithelial cells contributes to the pathogenesis of P. aeruginosa keratitis. To clarify the relationship between cell invasion and cytotoxicity, fourteen P. aeruginosa isolates were compared for their capacity to enter epithelial cells and for their ability to induce cytotoxicity. Bacterial invasion was quantified by gentamicin survival assays both in vivo and in vitro. Cytotoxicity was examined qualitatively by trypan blue exclusion assays and quantitatively by chromium release assays in vitro. A significant inverse correlation was found between the ability to induce cytotoxicity and epithelial cell invasion as measured by gentamicin survival assays. Both cytotoxic and noncytotoxic strains were identified among corneal and noncorneal isolates; all isolates that were not cytotoxic were capable of epithelial cell invasion. Efficient host cell invasion could not be demonstrated for cytotoxic strains; however, the gentamicin survival assay relies upon host cells retaining viability in order to yield useful results, and this may limit the effectiveness of this assay for testing epithelial cell invasion by cytotoxic strains. Since all of the corneal isolates that were tested were virulent in vivo, the results show that there are at least two different types of P. aeruginosa-induced disease, one caused by strains that are cytotoxic and the other involving bacteria that can enter epithelial cells and survive intracellularly without killing the host cell.

Contribution of proteases and LasR to the virulence of Pseudomonas aeruginosa during corneal infections.

The roles of the Pseudomonas aeruginosa proteases LasB (elastase) and LasA and the transcriptional activator LasR, which regulates the expression of these proteases, were evaluated in a murine model of P. aeruginosa corneal infection. In scarified corneas, P. aeruginosa PAO-A1 (LasA negative) or PAO-B1A1 (LasB and LasA negative) at a dose of 10(8) CFU per eye caused very mild or no disease following infection; however, the defect in PAO-A1 could not be complemented by supplying a functional copy of lasA either on a plasmid or inserted into the chromosome. In contrast, PAO-B1 (LasB negative) colonized the cornea and caused disease equal in severity to disease caused by the parental strain, PAO1-I. Although LasR is a known regulator of lasA expression, PAO-R1, a lasR-negative derivative of PAO1-I, was as virulent as the parental strain during corneal infection. When transcriptional fusion plasmids were used to quantify the expression of the lasB and lasA genes in P. aeruginosa PAO1-I and PAO-R1, the lasB::lacZ fusion in PAO-R1 showed only 3.5% as much activity as it did in PAO1-I, while the activity of the lasA::lacZ fusion in PAO-R1 was 27.8% of that in PAO1-I. Coadministration of 5 microg of purified LasA protease with PAO-A1 did not reconstitute a wild-type infection. This treatment produced an acute toxic reaction leading to prolonged eyelid closure without inflammatory destruction of the cornea that was similar to that observed when LasA was administered alone. These results indicate that insertional inactivation of lasA renders P. aeruginosa avirulent in a murine model of keratitis and that neither LasR nor elastase production is required for the establishment and maintenance of corneal infection. However, the lack of virulence of the LasA-deficient strains cannot be ascribed with certainty to the deficiency of LasA from the available data.

Rapid and sensitive method for evaluating Pseudomonas aeruginosa virulence factors during corneal infections in mice.

A murine corneal scratch model has been used extensively to study various aspects of the pathogenesis of Pseudomonas aeruginosa, a common etiologic agent of corneal infections. This model uses mild inhalation anesthetics which keep the animals immobile for a relatively short time and promote the interaction between the infecting organisms and the corneal wound. Under these circumstances, only a small number of P. aeruginosa isolates delivered at inocula of > 10(7) CFU are infectious. We determined that this model is useful for studying other P. aeruginosa strains given at lower doses if injectable anesthetics are administered prior to infection to keep the animals immobile for 15 to 30 min. Under these conditions, eight clinical isolates of P. aeruginosa tested at doses of 10(8) CFU per eye induced corneal perforation and/or phthisis in C3H/HeN mice. The 50% infective doses of several strains were between 3 x 10(2) and 1 x 10(5) CFU per mouse eye. When this modified anesthetic procedure was used to evaluate the roles of different P. aeruginosa virulence factors in eye infections, pathology was not observed when eyes were inoculated with 10(8) CFU of strains deficient in production of a complete lipopolysaccharide or the RpoN sigma factor. A strain with a point mutation in the fur gene, involved in production of iron-regulated factors, showed decreased virulence, while a mutant deficient in both hemolytic and nonhemolytic phospholipase C was fully virulent. By modifying the anesthesia procedure, the corneal scratch model allows rapid evaluations of the roles of P. aeruginosa virulence factors in corneal infections.