Lung Defense through IL-8 Carries a Cost of Chronic Lung Remodeling and Impaired Function.

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

Assessment of biofilm formation in Pseudomonas aeruginosa by antisense mazE-PNA.

The hallmark patogenicity in Pseudomonas aeruginosa (P. aeruginosa) is biofilm formation that is not easy to eradicate, because it has variety mechanisms for antibiotic resistance. In addition, toxin-antitoxin (TA) system may play role in biofilm formation. The current study aimed to evaluate the role of TA loci in biofilm formation. Therefore, 18 P. aeruginosa clinical isolates were collected and evaluated for specific biofilm and TA genes. The analysis by RT-qPCR demonstrated that expression of mazE antitoxin in biofilm formation was increase. On the other hand, mazE antitoxin TA system was used as target for antisense PNA. mazE-PNA was able to influence in biofilm formation and was inhibit at 5,10 and 15 µM concentrations biofilm formation in P. aeruginosa. Therefore, it could be highlighted target for anti-biofilm target to eradicate P. aeruginosa biofilm producer.

Assessment of IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis by an enzyme-linked immunosorbent assay (ELISA).

BACKGROUND: The usefulness of serological tests for detection of P. aeruginosa pulmonary infection in cystic fibrosis (CF) is controversial. Here, we assessed the value of detecting anti-P. aeruginosa IgG by a quantitative enzyme-linked immunosorbent assay (ELISA) for identification of P. aeruginosa infection in patients with cystic fibrosis. METHODS: Serum concentrations of anti-P. aeruginosa IgG were assessed in 117 CF patients classified according to their P. aeruginosa colonization/infection status (never colonized; free of infection; intermittently colonized and chronically infected) and in 53 healthy subjects by the ELISA test standardized with the St-Ag:1-17 antigen. RESULTS: The rate of IgG seropositivity and the median of IgG concentrations of this antibody in patients chronically infected were significantly higher than those found in the other CF groups and in the healthy control group. CONCLUSION: Detection of anti-P. aeruginosa IgG can be an useful tool for identification of P. aeruginosa chronic infection in patients with CF. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_158.

Assessment of panobacumab as adjunctive immunotherapy for the treatment of nosocomial Pseudomonas aeruginosa pneumonia.

The fully human anti-lipopolysaccharide (LPS) immunoglobulin M (IgM) monoclonal antibody panobacumab was developed as an adjunctive immunotherapy for the treatment of O11 serotype Pseudomonas aeruginosa infections. We evaluated the potential clinical efficacy of panobacumab in the treatment of nosocomial pneumonia. We performed a post-hoc analysis of a multicenter phase IIa trial (NCT00851435) designed to prospectively evaluate the safety and pharmacokinetics of panobacumab. Patients treated with panobacumab (n = 17), including 13 patients receiving the full treatment (three doses of 1.2 mg/kg), were compared to 14 patients who did not receive the antibody. Overall, the 17 patients receiving panobacumab were more ill. They were an average of 72 years old [interquartile range (IQR): 64-79] versus an average of 50 years old (IQR: 30-73) (p = 0.024) and had Acute Physiology and Chronic Health Evaluation II (APACHE II) scores of 17 (IQR: 16-22) versus 15 (IQR: 10-19) (p = 0.043). Adjunctive immunotherapy resulted in an improved clinical outcome in the group receiving the full three-course panobacumab treatment, with a resolution rate of 85 % (11/13) versus 64 % (9/14) (p = 0.048). The Kaplan-Meier survival curve showed a statistically significantly shorter time to clinical resolution in this group of patients (8.0 [IQR: 7.0-11.5] versus 18.5 [IQR: 8-30] days in those who did not receive the antibody; p = 0.004). Panobacumab adjunctive immunotherapy may improve clinical outcome in a shorter time if patients receive the full treatment (three doses). These preliminary results suggest that passive immunotherapy targeting LPS may be a complementary strategy for the treatment of nosocomial O11 P. aeruginosa pneumonia.

[Obtaining of outer membrane protein I of Pseudomonas aeruginosa and assessment of its antigenic properties].

Gene of outer membrane protein I (OprI) of Pseudomonas aeruginosa was cloned in Escherichia coli cells. Synthesized protein OprI contained additional sequence of 6 histidines on the N-terminus, which allowed its chromatographic purification in Ni-agarose. Obtained recombinant ptotein specifically reacted with hyperimmune rabbit serum against whole-cell P. aeruginosa and stimulated synthesis of specific antibodies in immunized mice and rabbits. Obtained hyperimmune rabbit sera against recombinant protein OprI had directive antimicrobial activity against P. aeruginosa.

Assessment of pulmonary antibodies with induced sputum and bronchoalveolar lavage induced by nasal vaccination against Pseudomonas aeruginosa: a clinical phase I/II study.

BACKGROUND: Vaccination against Pseudomonas aeruginosa is a desirable albeit challenging strategy for prevention of airway infection in patients with cystic fibrosis. We assessed the immunogenicity of a nasal vaccine based on the outer membrane proteins F and I from Pseudomonas aeruginosa in the lower airways in a phase I/II clinical trial. METHODS: N = 12 healthy volunteers received 2 nasal vaccinations with an OprF-OprI gel as a primary and a systemic (n = 6) or a nasal booster vaccination (n = 6). Antibodies were assessed in induced sputum (IS), bronchoalveolar lavage (BAL), and in serum. RESULTS: OprF-OprI-specific IgG and IgA antibodies were found in both BAL and IS at comparable rates, but differed in the predominant isotype. IgA antibodies in IS did not correlate to the respective serum levels. Pulmonary antibodies were detectable in all vaccinees even 1 year after the vaccination. The systemic booster group had higher IgG levels in serum. However, the nasal booster group had the better long-term response with bronchial antibodies of both isotypes. CONCLUSION: The nasal OprF-OprI-vaccine induces a lasting antibody response at both, systemic and airway mucosal site. IS is a feasible method to non-invasively assess bronchial antibodies. A further optimization of the vaccination schedule is warranted.

Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors.

CONTEXT: Patients with cystic fibrosis (CF) are susceptible to lower respiratory tract infections with Pseudomonas aeruginosa and typically acquire this organism in early childhood. Once P aeruginosa infection is established, eradication may be impossible, and progressive lung disease often aggravates morbidity and mortality risks. The ability to diagnose CF by genetic testing at birth makes it possible to determine the temporal sequence of events that result in P aeruginosa-associated pulmonary infections. OBJECTIVE: To evaluate the longitudinal relationship between the production of an antibody response against P aeruginosa and clinical factors associated with P aeruginosa pulmonary infections in patients with CF diagnosed in early life. DESIGN, SETTING, AND PATIENTS: Serum samples and oropharyngeal cultures (protocol cultures) were obtained at 6-month intervals from April 15, 1985, to April 15, 2000 (or for up to 180 months depending on their enrollment date) from 68 patients at 2 centers in Madison and Milwaukee, Wis, diagnosed through the Wisconsin CF Neonatal Screening Project, a longitudinal cohort study. Additional cultures were obtained at examining physicians' discretion (all cultures). MAIN OUTCOME MEASURES: Time to serum IgG, IgA, and IgM antibody titer of at least 1:256 against P aeruginosa, assessed by enzyme-linked immunosorbent assay using cell lysate, exotoxin A, and elastase as antigens; time to organism isolation from respiratory samples; time to Wisconsin Cystic Fibrosis Radiograph (WCXR) score of 5 or more. RESULTS: The median time to an antibody titer of at least 1:256 was 17.8, 24.2, and 70.9 months for cell lysate, exotoxin A, and elastase, respectively. The rise of anti-cell lysate and anti-exotoxin A titers to 1:256 or more occurred a mean of 11.9 (P<.001) and 5.6 (P =.04) months, respectively, before the isolation of P aeruginosa for all cultures and 18.2 (P<.001) and 11.9 (P =.006) months, respectively, before protocol cultures. There was no significant difference between the rise of anti-cell lysate and anti-exotoxin A titer and a WCXR score of 5 or more (P =.24 and.32, respectively). Treatment with long-term, non-Pseudomonas oral antibiotics and integration of CF infants with older, chronically infected patients were associated with a significantly increased risk of P aeruginosa pulmonary infection. CONCLUSIONS: In CF patients diagnosed through neonatal screening, P aeruginosa pulmonary infections occurred 6 to 12 months before the organism was isolated from respiratory secretions. The longitudinal monitoring of P aeruginosa antibody titers, in concert with WCXR score, should facilitate diagnosis and treatment of P aeruginosa pulmonary infections in young children with CF.

Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis.

Pseudomonas aeruginosa lung infection is an important cause of morbidity and mortality in cystic fibrosis (CF). Longitudinal assessment of the phenotypic changes in P. aeruginosa isolated from young children with CF is lacking. This study investigated genotypic and phenotypic changes in P. aeruginosa from oropharynx (OP) and bronchoalveolar lavage fluid (BALF) in a cohort of 40 CF patients during the first 3 years of life; antibody response was also examined. A high degree of genotypic variability was identified, and each patient had unique genotypes. Early isolates had a phenotype distinct from those of usual CF isolates: generally nonmucoid and antibiotic susceptible. Genotype and phenotype correlated between OP and BALF isolates. As determined by culture, 72.5% of patients demonstrated P. aeruginosa during their first 3 years. On the basis of combined culture and serologic results, 97.5% of patients had evidence of infection by age 3 years, which suggests that P. aeruginosa infection occurs early in CF and may be intermittent or undetectable by culture.

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: assessment of cross-reactivity with Pseudomonas aeruginosa.

Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa. The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa. The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. this study suggests that there is a specific anti-B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa. Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.

Assessment of biological activity of immunoglobulin preparations by using opsonized micro-organisms to stimulate neutrophil chemiluminescence.

We have used the ability of opsonised bacteria to stimulate luminol enhanced chemiluminescence of human neutrophils to examine the opsonic capabilities of normal and hypogammaglobulinaemic sera for four common bacterial pathogens. Preparations of human immunoglobulin modified for i.v. use have then been compared with unmodified Cohn Fraction II for their effectiveness in improving opsonization when added to antibody deficient sera in vitro. Hypogammaglobulinaemic sera exhibited impaired opsonisation of Haemophilus influenzae, and severely antibody deficient sera also opsonized Streptococcus pneumoniae and Pseudomonas aeruginosa poorly. The opsonization of these organisms was improved by Cohn Fraction II, and by pH 4 and beta-propionolactone treated immunoglobulins, in descending order of effectiveness. Pepsin digested immunoglobulin was inactive, and in some cases impaired opsonic capacity. The opsonisation of Staphylococcus aureus by hypogammaglobulinaemic sera was near normal, and was not improved by any immunoglobulin. This technique, which assesses biological activity of immunoglobulin, is useful in comparing preparations, and may help to establish appropriate dosage and frequency for intravenous immunoglobulin replacement therapy.