Elevated Peptides in Lung Lavage Fluid Associated with Bronchiolitis Obliterans Syndrome

<div><p>Objective</p><p>The objective of this discovery-level investigation was to use mass spectrometry to identify low mass compounds in bronchoalveolar lavage fluid from lung transplant recipients that associate with bronchiolitis obliterans syndrome.</p><p>Experimental Design</p><p>Bronchoalveolar lavage fluid samples from lung transplant recipients were evaluated for small molecules using ESI-TOF mass spectrometry and correlated to the development of bronchiolitis obliterans syndrome. Peptides associated with samples from persons with bronchiolitis obliterans syndrome and controls were identified separately by MS/MS analysis.</p><p>Results</p><p>The average bronchoalveolar lavage fluid MS spectrum profile of individuals that developed bronchiolitis obliterans syndrome differed greatly compared to controls. Controls demonstrated close inter-sample correlation (R = 0.97+/−0.02, average+/−SD) while bronchiolitis obliterans syndrome showed greater heterogeneity (R = 0.86+/−0.09, average+/−SD). We identified 89 features that were predictive of developing BOS grade 1 and 66 features predictive of developing BOS grade 2 or higher. Fractions from MS analysis were pooled and evaluated for peptide content. Nearly 10-fold more peptides were found in bronchiolitis obliterans syndrome relative to controls. C-terminal residues suggested trypsin-like specificity among controls compared to elastase-type enzymes among those with bronchiolitis obliterans syndrome.</p><p>Conclusions</p><p>Bronchoalveolar lavage fluid from individuals with bronchiolitis obliterans syndrome has an increase in low mass components detected by mass spectrometry. Many of these features were peptides that likely result from elevated neutrophil elastase activity.</p></div

C-Terminal amino acid comparison of unique endogenous peptides identified in BALF of transplant patients, control compared to BOS.

<p>C-Terminal amino acid comparison of unique endogenous peptides identified in BALF of transplant patients, control compared to BOS.</p

Features predictive of BOS.

<p>The median intensity of each feature is represented by the mass/charge (m/z) and retention time (time). Using a false discovery rate of 10%, the circles indicate features that are predictive of time to BOS 1 (triangles, n = 89) and those that are predictive of time to BOS 2 or 3 (circles, n = 66).</p

Top molecular and cellular biofunctions identified from protein substrates.

<p>Proteins identified from the sequenced peptides were used in Ingenuity Pathway Analysis to determine the molecular and cellular function of the substrate proteins. The number of proteins associated with each function is listed next to the bar.</p

UPLC separation of BALF fluid.

<p>Panel A. Total ion current for a typical sample from an individual with BOS. Panel B. Total ion current for a control subject who did not develop BOS within at least 100 months. The intensity has been adjusted to the same maximum as Panel A. The large peak at 1.48 minutes corresponded to lidocaine. Panel C. Extracted ion current for the +4 charge state of a component at m/z = 410.98. Only peak b represented a monoisotopic ion. Peaks a and c were isotopes of other compounds.</p

Titers of anti-PIV5 antibodies in the PIV5-vaccinated dogs.

<p>Eight dogs which had been vaccinated with live PIV5 were immunized with one dose of 8×10⁷ PFU of rPIV5-H3 viruses in 1 mL or PBS via intranasal route. The dogs were divided into two groups: two dogs received PBS; The remaining six dogs received rPIV5-H3. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and viral neutralization antibody assay (B). Data were presented as average value of duplicate wells. In the neutralization antibody assay, the white column indicates the PIV5 nAb titer is equal to or higher than 10.</p

Replication of PIV5 in dogs without PIV5 exposure.

<p>The nasal swabs of dogs were collected at 3 and 5 days post infection, and placed into a vial containing 0.5 mL of DMEM with 2% FBS. (A) Detection of virus with RT-PCR. (B) Detection of virus with plaque assay. Swab samples were examined by plaque assay on BHK21 cells. Two replicates for each serially diluted swab sample (1∶10⁰ to 1∶10²) were used in the assay.</p

Titers of anti-PIV5 antibodies in dogs without PIV5 exposure.

<p>Eight PIV5 naïve dogs were immunized with one dose of 8×10⁷ PFU of PIV5 or rPIV5-H3 viruses by intranasal route. The dogs were divided into two groups: PIV5-infected dogs and rPIV5-H3-infected dogs. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and virus neutralization antibody (nAb) assay (B). The grey columns indicate that the PIV5 nAb titer is less than 10, the limit of detection in this assay. The black columns indicate that the nAb titer is equal to or higher than 10.</p

PIV5 antibodies in humans.

<p>45 human serum samples were obtained from 18–50 year old healthy individuals. (A) Comparison of anti-PIV5 and anti-MuV antibody levels. ELISA was performed on plates coated with purified PIV5 or purified MuV with sera serially diluted. PIV5 or Mumps virus specific ELISA OD₄₅₀ values were shown at 320-fold dilution for each human serum sample. (B) Titers of neutralizing antibody against PIV5 in human sera. Data for the antibody titers were the average value of duplicate wells and presented for each human sample. The white column indicates that the PIV5 nAb titer is less than 10, the limit of detection. The black column indicates that the nAb titer is equal to or higher than 10.</p

Replication of PIV5 in dogs with prior PIV5 vaccination.

<p>The nasal swabs of dogs were collected at 3 and 5 dpi. Detections of virus were performed the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050144#pone-0050144-g002" target="_blank">Fig. 2</a>. (A) RT-PCR and (B) Plaque assay.</p