BackgroundIdiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues.ResultsWe isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four.ConclusionsOur results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-016-0356-4) contains supplementary material, which is available to authorized users.

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Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis.

Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen α2(I) mRNA was at normal levels. Collagen α1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into α1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis.

This report confirms the gram-negative ultrastructural characteristics of the Legionnaires' disease organism by direct examination of pulmonary tissue from six confirmed cases--two from the original Philadelphia epidemic of 1976 and four from more recent sporadic cases. All microorganisms seen in all six lungs were identical ultrastructurally and were predominantely within intra-alveolar macrophages, as previously observed by light microscopy. They appeared as short, blunt rods that were clearly prokaryotic; i.e., they had diffuse electron-lucent nucleoid areas interspersed among areas of well-defined ribosomes, a pinching nonseptic division, and enclosure within a double envelope consisting of two three-layer "unit" membranes, each approximately 75 A wide. This structure, together with a pinching division, is typical of gram-negative bacteria. The Legionnaires' disease organism multiples both intracellularly and extracellularly in tissue and has no unique ultrastructural features that would aid in its specific identification. These findings are compared with recent reports describing the ultrastructure of what was considered to be the Legionnaires' disease organism in yolk sac and culture medium, and in one human lung.

Patients with acute Legionnaires' disease (LD) pneumonia may have persistent chronic pulmonary changes, as shown by the histologic appearance of specimens of lung from patients who had survived and autopsy specimens from patients who died after a protracted clinical course. Acute pneumonia was not seen in these lungs, and LD organisms could not be identified by the direct fluorescent antibody technique or the Dieterle silver impregnation strain; instead, there was organizing pneumonia with various degrees of interstitial inflammation and fibrosis. The LD pneumonia may fail to resolve, and the lung parenchyma in areas of previous acute inflammation is not restored to normal in some patients.

Immunofluorescent study of the lungs in cases of fatal suspected acute Legionnaires' disease enabled confirmation of the presence of Legionella pneumophila. In addition, probable pathogenetic mechanisms that had not been as clearly visualized by light microscopy became apparent: the retrograde involvement of the larger bronchioles and proximal airways, invasion of the interstitium, extension to pleura, and lymphatic and hematogenous spread. Organisms were demonstrated to occur in the liver and spleen of one patient. The development of technics for the earliest possible diagnostic verification of Legionnaires' disease, with specimens obtained as untraumatically as practical from selected sites and screened by specific immunofluorescent microscopic examination, should contribute to patient survival.

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