Quantification of Cytokeratin 5 mRNA Expression in the Circulation of Healthy Human Subjects and after Lung Transplantation

Circulating epithelial progenitor cells are important for repair of the airway epithelium in a mouse model of tracheal transplantation. We therefore hypothesized that circulating epithelial progenitor cells would also be present in normal human subjects and could be important for repair of the airway after lung injury. As lung transplantation is associated with lung injury, which is severe early on and exacerbated during episodes of infection and rejection, we hypothesized that circulating epithelial progenitor cell levels could predict clinical outcome following lung transplantation.Quantitative Real Time PCR was performed to determine peripheral blood mRNA levels of cytokeratin 5, a previously characterized marker of circulating epithelial progenitor cells. Cytokeratin 5 levels were evaluated in healthy human subjects, in lung transplant recipients immediately post-transplant and serially thereafter, and in heart transplant recipients. All normal human subjects examined expressed cytokeratin 5 in their buffy coat in amounts that were not significantly influenced by age or gender. There was a profound, statistically significant decrease in cytokeratin 5 mRNA expression levels in lung transplant patients compared to healthy human subjects (p = 3.1x10(-13)) and to heart transplant recipients. There was a moderate negative correlation between improved circulating cytokeratin 5 mRNA levels in lung transplant recipients with recovering lung function, as measured by improved FEV1 values (rho = -0.39).Levels of cytokeratin 5 mRNA, a proxy marker for circulating epithelial progenitor cells, inversely correlated with disease status in lung transplant recipients. It may therefore serve as a biomarker of the clinical outcome of lung transplant patients and potentially other patients with airway injury

Immune response CC chemokines CCL2 and CCL5 are associated with pulmonary sarcoidosis

Abstract Background Pulmonary sarcoidosis involves an intense leukocyte infiltration of the lung with the formation of non-necrotizing granulomas. CC chemokines (chemokine (C-C motif) ligand 2 (CCL2)-CCL5) are chemoattractants of mononuclear cells and act through seven transmembrane G-coupled receptors. Previous studies have demonstrated conflicting results with regard to the associations of these chemokines with sarcoidosis. In an effort to clarify previous discrepancies, we performed the largest observational study to date of CC chemokines in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis. Results BALF chemokine levels from 72 patients affected by pulmonary sarcoidosis were analyzed by enzyme-linked immunosorbent assay (ELISA) and compared to 8 healthy volunteers. BALF CCL3 and CCL4 levels from pulmonary sarcoidosis patients were not increased compared to controls. However, CCL2 and CCL5 levels were elevated, and subgroup analysis showed higher levels of both chemokines in all stages of pulmonary sarcoidosis. CCL2, CCL5, CC chemokine receptor type 1 (CCR1), CCR2 and CCR3 were expressed from mononuclear cells forming the lung granulomas, while CCR5 was only found on mast cells. Conclusions These data suggest that CCL2 and CCL5 are important mediators in recruiting CCR1, CCR2, and CCR3 expressing mononuclear cells as well as CCR5-expressing mast cells during all stages of pulmonary sarcoidosis

Capillary Proliferation in Systemic-Sclerosis-Related Pulmonary Fibrosis: Association with Pulmonary Hypertension

We sought to determine if any histopathologic component of the pulmonary microcirculation can distinguish systemic sclerosis (SSc)-related pulmonary fibrosis (PF) with and without pulmonary hypertension (PH)

Quantification of cytokeratin 5 mRNA expression in the circulation of healthy human subjects and after lung transplantation.

BackgroundCirculating epithelial progenitor cells are important for repair of the airway epithelium in a mouse model of tracheal transplantation. We therefore hypothesized that circulating epithelial progenitor cells would also be present in normal human subjects and could be important for repair of the airway after lung injury. As lung transplantation is associated with lung injury, which is severe early on and exacerbated during episodes of infection and rejection, we hypothesized that circulating epithelial progenitor cell levels could predict clinical outcome following lung transplantation.Methodology/principal findingsQuantitative Real Time PCR was performed to determine peripheral blood mRNA levels of cytokeratin 5, a previously characterized marker of circulating epithelial progenitor cells. Cytokeratin 5 levels were evaluated in healthy human subjects, in lung transplant recipients immediately post-transplant and serially thereafter, and in heart transplant recipients. All normal human subjects examined expressed cytokeratin 5 in their buffy coat in amounts that were not significantly influenced by age or gender. There was a profound, statistically significant decrease in cytokeratin 5 mRNA expression levels in lung transplant patients compared to healthy human subjects (p = 3.1x10(-13)) and to heart transplant recipients. There was a moderate negative correlation between improved circulating cytokeratin 5 mRNA levels in lung transplant recipients with recovering lung function, as measured by improved FEV1 values (rho = -0.39).Conclusions/significanceLevels of cytokeratin 5 mRNA, a proxy marker for circulating epithelial progenitor cells, inversely correlated with disease status in lung transplant recipients. It may therefore serve as a biomarker of the clinical outcome of lung transplant patients and potentially other patients with airway injury

Figure 2

<p>A. Quantitative real-time PCR of CK5 plotted against age of normal human subjects. A scatter plot of data shows no significant difference between the log of the age and CK5 mRNA expression levels in all 38 normal human subjects examined (p = 0.273). B. Quantitative real-time PCR expression of CK5 does not differ with gender. A box plot demonstrates the similarity in CK5 levels in male and female normal human subjects (p = 0.84).</p

Optimization of the Assay for Primers and Probes.

<p>The Quantitative Real Time PCR assay was first performed with varying concentrations of primer and then probe to determine the optimal concentration of primers and probe for the assay. The concentration at which, for both primers and probe, the ΔCt and ΔRn did not change with increasing concentrations was selected. Thus primer concentrations of 600 nM and probe concentration of 250 nM were chosen.</p

Figure 4

<p>A. Quantitative real-time PCR of CK5 expression in lung transplant patients correlated with time post-transplant. This box plot shows the increase in CK5 mRNA expression (decrease in ΔCt for CK5) with time post transplant. The X-axis represents set time points post transplant (0 = time of transplant; 1 = 1day post-transplant (PT); 2 = 1week PT; 3 = 1month PT; 4 = 3months PT; 5 = 6months PT). B. Percentage decrease in FEV1 in lung transplant patients correlated with time post-transplant. Box plot of FEV1 on the Y-axis with 0.0 being 100% FEV1 compared to time post transplant on the X-axis (0 = time of transplant; 1 = 1day PT; 2 = 1week PT; 3 = 1month PT; 4 = 3months PT; 5 = 6months PT). C. Quantitative real-time PCR of CK5 expression in lung transplant patients correlated with percentage decrease in FEV1 post-transplant. There is a moderate negative correlation between average FEV1 and average ΔCt values of −0.39. However, this does not reach statistical significance with a p-value = 0.085.</p

Lung transplant patient demographics.

<p>COPD = chronic obstructive pulmonary disease; IPF = idiopathic pulmonary fibrosis; PHTN = pulmonary hypertension; ILD = interstitial lung disease; PCH = Pulmonary capillary hemangiomatosis.</p

Figure 1

<p>A. PCR for CK5 mRNA from the circulation of healthy volunteers and lung transplant patients. The top panel shows the expected 439 bp fragment for CK5 using cDNA as template in healthy volunteers (Lanes 1–4) and CK5 mRNA expression from a representative group of patients post lung transplantation (Lanes 5–9). CK5 mRNA expression was not found in PCR Lanes 5, 8 and 9 and neither was CK5 mRNA expression detectible by quantitative real-time PCR in these samples. Lane 10 represents the positive control, which consists of a bacterial artificial chromosome (BAC) containing 170 kb of genomic sequence surrounding the CK5 locus as template, which is why the band is a larger size. Lane 11 is the negative control without cDNA template. The bottom panel shows PCR amplification of GAPDH from the same templates. B. Standard curve of real-time PCR amplification of CK5 and GAPDH. The log quantity of RNA is plotted against the mean threshold cycle (Ct), measured in triplicate. Slope and intercept are represented in the equations of the regression lines, along with the regression coefficient.</p