Effect of bronchoalveolar lavage-directed therapy on pseudomonas aeruginosa infection and structural lung injury in children with cystic fibrosis

Context Early pulmonary infection in children with cystic fibrosis leads to increased morbidity and mortality. Despite wide use of oropharyngeal cultures to identify pulmonary infection, concerns remain over their diagnostic accuracy. While bronchoalveolar lavage (BAL) is an alternative diagnostic tool, evidence for its clinical benefit is lacking.

Objective To determine if BAL-directed therapy for pulmonary exacerbations during the first 5 years of life provides better outcomes than current standard practice relying on clinical features and oropharyngeal cultures.

Design, Setting, and Participants The Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) randomized controlled trial, recruiting infants diagnosed with cystic fibrosis through newborn screening programs in 8 Australasian cystic fibrosis centers. Recruitment occurred between June 1, 1999, and April 30, 2005, with the study ending on December 31, 2009.

Interventions BAL-directed (n=84) or standard (n=86) therapy until age 5 years. The BAL-directed therapy group underwent BAL before age 6 months when well, when hospitalized for pulmonary exacerbations, if Pseudomonas aeruginosa was detected in oropharyngeal specimens, and after P aeruginosa eradication therapy. Treatment was prescribed according to BAL or oropharyngeal culture results.

Main Outcome Measures Primary outcomes at age 5 years were prevalence of P aeruginosa on BAL cultures and total cystic fibrosis computed tomography (CF-CT) score (as a percentage of the maximum score) on high-resolution chest CT scan.

Results Of 267 infants diagnosed with cystic fibrosis following newborn screening, 170 were enrolled and randomized, and 157 completed the study. At age 5 years, 8 of 79 children (10%) in the BAL-directed therapy group and 9 of 76 (12%) in the standard therapy group had P aeruginosa in final BAL cultures (risk difference, −1.7% [95% confidence interval, −11.6% to 8.1%]; P=.73). Mean total CF-CT scores for the BAL-directed therapy and standard therapy groups were 3.0% and 2.8%, respectively (mean difference, 0.19% [95% confidence interval, −0.94% to 1.33%]; P=.74).

Conclusion Among infants diagnosed with cystic fibrosis, BAL-directed therapy did not result in a lower prevalence of P aeruginosa infection or lower total CF-CT score when compared with standard therapy at age 5 years.

Trial Registration anzctr.org.au Identifier: ACTRN12605000665639

Costs of bronchoalveolar lavage-directed therapy in the first 5 years of life for children with cystic fibrosis.

To determine whether bronchoalveolar lavage (BAL)-directed therapy for infants and young children with cystic fibrosis (CF), rather than standard therapy, was justified on the grounds of a decrease in average costs and whether the use of BAL reduced treatment costs associated with hospital admissions.

Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic airway disease in mice

Background: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases.

Objective: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses.

Methods: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge.

Results: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective.

Conclusion: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.

Long lived multi-isotype anti-HIV antibody responses following a prime-double boost immunization strategy

Despite decades of work, an effective HIV vaccine remains elusive. In an effort to elicit protective immunity, investigators have sought to define vaccines able to elicit durable HIV-specific B-cell and T-cell activities. Additionally, vaccines are sought which can induce antibodies of a variety of isotypes, as each isotype possesses unique attributes in terms of opsonization, Fc receptor binding capacity, complement fixation and location. One prominent new vaccine strategy, applied to numerous distinct antigenic systems is the prime boost-regimen, with DNA, vaccinia virus (VV), and/or purified recombinant protein. To examine the durability, location and isotype distribution of responses induced by prime-boost regimens, we tested successive immunizations with DNA, VV and protein (D-V-P), comparing three forms of protein inoculations: (i) purified protein administered intramuscularly with complete Freunds adjuvant, (ii) purified protein administered intranasally, and (iii) purified protein conjugated to oxidized mannan, administered intranasally. We found that all three protocols elicited serum antibodies of multiple isotypes, with serum IgA being most prominent among mice immunized with mannan-conjugated protein. All D-V-P protocols, regardless of protein form or route, also elicited antibody responses at mucosal surfaces. In bronchoalveolar lavage, a tendency toward IgA production was again most prominent in mice boosted with the protein–mannan conjugate. Both B-cell and T-cell responses were sustained for more than 1 year post-immunization following each form of vaccination. Contemporaneous with long-lasting serum and mucosal antibodies were antibody forming cells in the bone marrow of primed animals. Results highlight the D-V-P vaccination strategy as a promising approach for attaining durable, multi-isotype B-cell and T-cell activities toward HIV.

Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective: Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.
Methods: We measured NK and NKT-like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex-smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF.
Results: In blood in COPD, there were no significant changes in the proportion of NK or NKT-like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT-like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types.
Conclusions: Treatment strategies that target NK and NKT-like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity.

Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine

Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)–derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, FcγR1), M2 markers (dendritic cell–specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen–presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.

Prospective evaluation of respiratory exacerbations in children with cystic fibrosis from newborn screening to 5 years of age

Background Newborn screening allows novel treatments for cystic fibrosis (CF) to be trialled in early childhood before irreversible lung injury occurs. As respiratory exacerbations are a potential trial outcome variable, we determined their rate, duration and clinical features in preschool children with CF; and whether they were associated with growth, lung structure and function at age 5 years. Methods: Respiratory exacerbations were recorded prospectively in Australasian CF Bronchoalveolar Lavage trial subjects from enrolment after newborn screening to age 5 years, when all participants underwent clinical assessment, chest CT scans and spirometry. Results 168 children (88 boys) experienced 2080 exacerbations, at an average rate of 3.66 exacerbations per person-year; 80.1% were community managed and 19.9% required hospital admission. There was an average increase in exacerbation rate of 9% (95% CI 4% to 14%; p<0.001) per year of age. Exacerbation rate differed by site (p<0.001) and was 26% lower (95% CI 12% to 38%) in children receiving 12 months of prophylactic antibiotics. The rate of exacerbations in the first 2 years was associated with reduced forced expiratory volume in 1 s z scores. Ever having a hospitalmanaged exacerbation was associated with bronchiectasis (OR 2.67, 95% CI 1.13 to 6.31) in chest CT scans, and lower weight z scores at 5 years of age (coefficient -0.39, 95% CI -0.74 to -0.05). Conclusions Respiratory exacerbations in young children are markers for progressive CF lung disease and are potential trial outcome measures for novel treatments in this age group.

Zinc and zinc transporters in macrophages and their roles in efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.

Pseudomonas aeruginosa genotypes acquired by children with cystic fibrosis by age 5-years

BACKGROUND: We describe Pseudomonas aeruginosa acquisitions in children with cystic fibrosis (CF) aged ≤5-years, eradication treatment efficacy, and genotypic relationships between upper and lower airway isolates and strains from non-CF sources. METHODS: Of 168 CF children aged ≤5-years in a bronchoalveolar lavage (BAL)-directed therapy trial, 155 had detailed microbiological results. Overall, 201/271 (74%) P. aeruginosa isolates from BAL and oropharyngeal cultures were available for genotyping, including those collected before and after eradication therapy. RESULTS: Eighty-two (53%) subjects acquired P. aeruginosa, of which most were unique strains. Initial eradication success rate was 90%, but 36 (44%) reacquired P. aeruginosa, with genotypic substitutions more common in BAL (12/14) than oropharyngeal (3/11) cultures. Moreover, oropharyngeal cultures did not predict BAL genotypes reliably. CONCLUSIONS: CF children acquire environmental P. aeruginosa strains frequently. However, discordance between BAL and oropharyngeal strains raises questions over upper airway reservoirs and how to best determine eradication in non-expectorating children.

Pulse-lavage brushing followed by hydrogen peroxide-gauze packing for bone-bed preparation in cemented total hip arthroplasty : a bovine model

To compare the effectiveness of pulse-lavage brushing followed by hydrogen peroxide-gauze packing with either technique alone or normal-saline irrigation in bone-bed preparation for cemented total hip arthroplasty. 44 fresh-frozen ox femoral canals were prepared for cemented total hip arthroplasty using 4 techniques: normal-saline irrigation, pulse-lavage brushing, hydrogen peroxide-soaked gauze packing, and a combination of the latter 2 techniques. The maximum tensile pull-out force required to separate the prosthesis from the femoral canal was measured as an indicator of the strength of the cement-bone interface. The mean pull-out force to separate the prosthesis from the femoral canal was significantly higher in specimens prepared with pulse-lavage brushing followed by hydrogen peroxide-soaked gauze packing or pulse-lavage brushing alone than those prepared with normal-saline irrigation or hydrogen peroxide-soaked gauze packing alone 300(p<0.001). Pulse-lavage brushing is more effective at cleansing the femoral canal and increasing mechanical strength at the cement-bone interface than preparation with normal-saline irrigation or hydrogen peroxide-soaked gauze packing.

Developing a placebo-controlled trial in surgery : issues of design, acceptability and feasibility

Background: Surgical placebos are controversial. This in-depth study explored the design, acceptability, and feasibility issues relevant to designing a surgical placebo-controlled trial for the evaluation of the clinical and cost effectiveness of arthroscopic lavage for the management of people with osteoarthritis of the knee in the UK.
Methods: Two surgeon focus groups at a UK national meeting for orthopaedic surgeons and one regional surgeon focus group (41 surgeons); plenary discussion at a UK national meeting for orthopaedic anaesthetists (130 anaesthetists); three focus groups with anaesthetists (one national, two regional; 58 anaesthetists); two focus groups with members of the patient organisation Arthritis Care (7 participants); telephone interviews with people on consultant waiting lists from two UK regional centres (15 participants); interviews with Chairs of UK ethics committees (6 individuals); postal surveys of members of the British Association of Surgeons of the Knee (382 surgeons) and members of the British Society of Orthopaedic Anaesthetists (398 anaesthetists); two centre pilot (49 patients assessed).
Results: There was widespread acceptance that evaluation of arthroscopic lavage had to be conducted with a placebo control if scientific rigour was not to be compromised. The choice of placebo surgical procedure (three small incisions) proved easier than the method of anaesthesia (general anaesthesia). General anaesthesia, while an excellent mimic, was more intrusive and raised concerns among some stakeholders and caused extensive discussion with local decision-makers when seeking formal approval for the pilot. Patients were willing to participate in a pilot with a placebo arm; although some patients when allocated to surgery became apprehensive about the possibility of receiving placebo, and withdrew. Placebo surgery was undertaken successfully.
Conclusions: Our study illustrated the opposing and often strongly held opinions about surgical placebos, the ethical issues underpinning this controversy, and the challenges that exist even when ethics committee approval has been granted. It showed that a placebo-controlled trial could be conducted in principle, albeit with difficulty. It also highlighted that not only does a placebo-controlled trial in surgery have to be ethically and scientifically acceptable but that it also must be a feasible course of action. The place of placebo-controlled surgical trials more generally is likely to be limited and require specific circumstances to be met. Suggested criteria are presented.

Recombinant influenza viruses as a vaccine vector for HIV

 Live recombinant influenza viruses were successfully used as HIV vaccine vectors in a mouse model. Following intranasal prime-boost vaccination, HIV-specific CD8+ T cell responses were detected in the spleen, broncho-alveolar lavage, mediastinal and inguinal lymph nodes. HIV+α4β7+ CD8+ T cells contributed to protection in pseudo-challenge experiments using recombinant vaccinia virus expressing HIV antigens. This research highlights the importance of mucosal CD8+ T cells in viral immunity and emphasizes the need for additional studies to provide key insights to underpin future vaccine development.