Human subjects.

<p>Human subjects.</p

Methylation profile of LINE-1 retrotransposon.

<p>Global methylation levels of the LINE-1 retrotransposons were defined using the EpiTYPER mass array assay. Each bar represents the methylation in one of the 15 CpG dinucleotides or CpG units that span the LINE-1 sequence. Methylation levels are calculated as the average of all samples in each group (Control, IPF, Cancer) and standard error bars are included. The X axis shows the CpG dinucleotide or the CpG unit and the Y axis shows the percentage of methylation. The LINE-1 promoter is indicated in purple. The red arrowheads indicate units of 2-3 CpG dinucleotides.</p

Comparison of IPF and Adenocarcinoma to control samples.

<p>(A) 3-D plot representation of the results after Principal Component Analysis of all 3 sample groups. Each color-coded dot represents a sample (red-control, green-IPF and blue-cancer) and each sample is positioned in the 3-D space according to its similarity or difference to the others. (B) Comparison of differentially methylated CpG islands that overlap between IPF and Lung Cancer. The heatmap consists of 402 differentially methylated CpG islands that are found to overlap between IPF and Lung Cancer. High methylation levels of a CpG islands are shown in yellow while low methylation levels of methylation are shown in purple. Grey stands for no difference between the two groups being compared.</p

Expression of genes with differentially methylated promoters.

<p>qRT-PCR assay on 3 genes with hypomethylated promoter-associated CpG islands showed increase in the expression of the downstream gene. Y-axis shows fold change of detected transcripts in IPF samples when the expression in controls is set to baseline equal to 1. * denotes p-values <0.05. Error bars are based on Standard Deviation.</p

CpG islands are differentially methylated in IPF and control samples

<p>(A) Human CpG Island Microarray data: the heatmap on the left is the visual comparison of global methylation profiles between the 10 Control and the 12 IPF samples. The heatmap on the right consists only of the differentially methylated CpG probes highlighted by the red rectangle (<i>p</i>-value < 0.05, FDR<5%). Methylated CpG islands are shown in progressively brighter shades of yellow, depending on the fold difference, and hypomethylated CpG islands are shown in progressively brighter shades of purple. Grey stands for no difference between the two sample groups being compared. (B) EpiTYPER confirmation of differentially methylated CpG islands. Bars represent the average methylation of all the samples per study group. The X-axis shows the genomic location of each CpG island and Y-axis shows the percentage of methylation.</p

Differentially methylated promoters between IPF and control samples.

<p>Differentially methylated promoters between IPF and control samples.</p

Functional annotation clustering of differentially methylated CpG islands.

<p>Functional annotation clustering of differentially methylated CpG islands.</p

Autoantibody-Targeted Treatments for Acute Exacerbations of Idiopathic Pulmonary Fibrosis

<div><p>Background</p><p>Severe acute exacerbations (AE) of idiopathic pulmonary fibrosis (IPF) are medically untreatable and often fatal within days. Recent evidence suggests autoantibodies may be involved in IPF progression. Autoantibody-mediated lung diseases are typically refractory to glucocorticoids and nonspecific medications, but frequently respond to focused autoantibody reduction treatments. We conducted a pilot trial to test the hypothesis that autoantibody-targeted therapies may also benefit AE-IPF patients.</p><p>Methods</p><p>Eleven (11) critically-ill AE-IPF patients with no evidence of conventional autoimmune diseases were treated with therapeutic plasma exchanges (TPE) and rituximab, supplemented in later cases with intravenous immunoglobulin (IVIG). Plasma anti-epithelial (HEp-2) autoantibodies and matrix metalloproteinase-7 (MMP7) were evaluated by indirect immunofluorescence and ELISA, respectively. Outcomes among the trial subjects were compared to those of 20 historical control AE-IPF patients treated with conventional glucocorticoid therapy prior to this experimental trial.</p><p>Results</p><p>Nine (9) trial subjects (82%) had improvements of pulmonary gas exchange after treatment, compared to one (5%) historical control. Two of the three trial subjects who relapsed after only five TPE responded again with additional TPE. The three latest subjects who responded to an augmented regimen of nine TPE plus rituximab plus IVIG have had sustained responses without relapses after 96-to-237 days. Anti-HEp-2 autoantibodies were present in trial subjects prior to therapy, and were reduced by TPE among those who responded to treatment. Conversely, plasma MMP7 levels were not systematically affected by therapy nor correlated with clinical responses. One-year survival of trial subjects was 46+15% vs. 0% among historical controls. No serious adverse events were attributable to the experimental medications.</p><p>Conclusion</p><p>This pilot trial indicates specific treatments that reduce autoantibodies might benefit some severely-ill AE-IPF patients. These findings have potential implications regarding mechanisms of IPF progression, and justify considerations for incremental trials of autoantibody-targeted therapies in AE-IPF patients.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/results?term=NCT01266317" target="_blank">NCT01266317</a></p></div

Survival Comparisons.

<p>A.) Survival in the aggregate trial subject population (n = 11) was greater than among historical control AE-IPF patients (n = 20). Cross hatches and numbers in parentheses denote censored observations. Lung transplantation censoring is denoted with “T”. B.) Clinical responses may be more durable and survival may be further enhanced among the later trial subjects (n = 4) treated with a more aggressive regimen of autoantibody-targeted modalities (9 initial TPE + rituximab + IVIG).</p

Flowchart of subject recruitments for these experimental trials.

<p>Original Regimen denotes the first series of subjects who were treated with the initial, relatively more conservative regimen (#1–7). Augmented Regimen denotes the most recent four subjects (#8–11) who received a more aggressive therapeutic course, based on interval results in the first cohort (see text for details). TPE = therapeutic plasma exchange; IVIG = intravenous immunoglobulin; * denotes oral consent of patients, under auspices of innovative clinical practice, that were given by these patients after being fully informed of potential risks and yet-unproven efficiencies of the novel treatments.</p