Kristen L. Veraldi

hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells.

ABSTRACT Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued fortrans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H′ was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H′, and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.

A Peptide Derived from Endostatin Ameliorates Organ Fibrosis

A naturally occurring peptide from endostatin can inhibit fibrosis in lung and skin, even when it is already established. Putting an End to Fibrotic Organ Failure with Endostatin? Organ fibrosis or “scarring” has the dubious distinction of accounting for nearly half of all deaths in the developed world, taking its toll in disorders ranging from liver cirrhosis to cardiovascular disease. There are no effective treatments for fibrosis, an out-of-control wound-healing process in which excess extracellular matrix components such as collagen and fibronectin replace normal tissue, ultimately resulting in organ failure. Endostatin, a naturally occurring proteolytic fragment of one form of collagen, is elevated in patients with different forms of fibrosis. Previously, endostatin was shown to inhibit the growth of new blood vessels, an effect attributed to its N-terminal region. Because signaling molecules involved in blood vessel formation are also present in fibrotic tissues, Yamaguchi et al. investigated whether endostatin might inhibit fibrosis as well. Indeed, they found that a recombinant form did inhibit dermal fibrosis, in this case induced by the profibrotic factor transforming growth factor–β (TGF-β) in an ex vivo human skin model. So, too, could a smaller peptide, termed E4, which was derived from the C terminus of endostatin. In addition, E4 prevented TGF-β–induced dermal fibrosis in a mouse model—as well as dermal and lung fibrosis induced by a different trigger, bleomycin. E4 also reduced bleomycin-induced cell death in the mouse lung. Furthermore, Yamaguchi et al. found that E4 could reverse ongoing fibrosis, in addition to preventing its initiation, in these models. Finally, the researchers showed that E4 reduced the expression of two key proteins: lysyl oxidase, an enzyme that cross-links collagen and thereby contributes to fibrosis, and the transcription factor Egr-1, which is a central fibrosis regulator. Because E4 can inhibit both skin and lung fibrosis, it may well be able to inhibit fibrosis in other tissues as well. Further research is needed to explore the therapeutic potential of this peptide for preventing or reversing organ fibrosis. Fibroproliferative disorders such as idiopathic pulmonary fibrosis and systemic sclerosis have no effective therapies and result in significant morbidity and mortality due to progressive organ fibrosis. We examined the effect of peptides derived from endostatin on existing fibrosis and fibrosis triggered by two potent mediators, transforming growth factor–β (TGF-β) and bleomycin, in human and mouse tissues in vitro, ex vivo, and in vivo. We identified one peptide, E4, with potent antifibrotic activity. E4 prevented TGF-β–induced dermal fibrosis in vivo in a mouse model, ex vivo in human skin, and in bleomycin-induced dermal and pulmonary fibrosis in vivo, demonstrating that E4 exerts potent antifibrotic effects. In addition, E4 significantly reduced existing fibrosis in these preclinical models. E4 amelioration of fibrosis was accompanied by reduced cell apoptosis and lower levels of lysyl oxidase, an enzyme that cross-links collagen, and Egr-1 (early growth response gene–1), a transcription factor that mediates the effects of several fibrotic triggers. Our findings identify E4 as a peptide with potent antifibrotic activity and a possible therapeutic agent for organ fibrosis.

Respiratory Bronchiolitis-Interstitial Lung Disease

Background:The clinical and physiologic features of respiratory bronchiolitis (RB)-interstitial lung disease (ILD) have been previously described; however, the natural history and outcome have not been systematically evaluated. The majority of published reports consider RB-ILD to be a nonprogressive ILD that clinically improves with smoking cessation and antiinflammatory treatment. In this study, we sought to determine the outcome of RB-ILD patients with and without smoking cessation and with and without corticosteroid therapy. Methods:Thirty-two RB-ILD cases confirmed by surgical lung biopsy were identified from a prospectively enrolled cohort of subjects with ILD. Initial and follow-up data on symptoms, physiology, treatment, and outcome were collected and analyzed. Results:Kaplan-Meier analysis revealed that at least 75% of RB-ILD patients survived > 7 years after diagnosis. Clinical improvement occurred in only 28% of cases, and physiologic improvement occurred in 10.5% of cases. One patient died of progressive ILD, and two patients died of non-small cell lung cancer. While physiologic improvement was limited to those who had ceased smoking, corticosteroids and/or other immunosuppressive therapy had little effect on symptoms or physiology. Conclusions:This study shows that prolonged survival is common in RB-ILD. However, symptomatic and physiologic improvement occurs in only a minority of patients, and neither smoking cessation nor immunosuppressive therapy is regularly associated with clinically significant benefit.BACKGROUND The clinical and physiologic features of respiratory bronchiolitis (RB)-interstitial lung disease (ILD) have been previously described; however, the natural history and outcome have not been systematically evaluated. The majority of published reports consider RB-ILD to be a nonprogressive ILD that clinically improves with smoking cessation and antiinflammatory treatment. In this study, we sought to determine the outcome of RB-ILD patients with and without smoking cessation and with and without corticosteroid therapy. METHODS Thirty-two RB-ILD cases confirmed by surgical lung biopsy were identified from a prospectively enrolled cohort of subjects with ILD. Initial and follow-up data on symptoms, physiology, treatment, and outcome were collected and analyzed. RESULTS Kaplan-Meier analysis revealed that at least 75% of RB-ILD patients survived > 7 years after diagnosis. Clinical improvement occurred in only 28% of cases, and physiologic improvement occurred in 10.5% of cases. One patient died of progressive ILD, and two patients died of non-small cell lung cancer. While physiologic improvement was limited to those who had ceased smoking, corticosteroids and/or other immunosuppressive therapy had little effect on symptoms or physiology. CONCLUSIONS This study shows that prolonged survival is common in RB-ILD. However, symptomatic and physiologic improvement occurs in only a minority of patients, and neither smoking cessation nor immunosuppressive therapy is regularly associated with clinically significant benefit.

Original Research: Interstitial Lung DiseaseRespiratory Bronchiolitis-Interstitial Lung Disease

Background:The clinical and physiologic features of respiratory bronchiolitis (RB)-interstitial lung disease (ILD) have been previously described; however, the natural history and outcome have not been systematically evaluated. The majority of published reports consider RB-ILD to be a nonprogressive ILD that clinically improves with smoking cessation and antiinflammatory treatment. In this study, we sought to determine the outcome of RB-ILD patients with and without smoking cessation and with and without corticosteroid therapy. Methods:Thirty-two RB-ILD cases confirmed by surgical lung biopsy were identified from a prospectively enrolled cohort of subjects with ILD. Initial and follow-up data on symptoms, physiology, treatment, and outcome were collected and analyzed. Results:Kaplan-Meier analysis revealed that at least 75% of RB-ILD patients survived > 7 years after diagnosis. Clinical improvement occurred in only 28% of cases, and physiologic improvement occurred in 10.5% of cases. One patient died of progressive ILD, and two patients died of non-small cell lung cancer. While physiologic improvement was limited to those who had ceased smoking, corticosteroids and/or other immunosuppressive therapy had little effect on symptoms or physiology. Conclusions:This study shows that prolonged survival is common in RB-ILD. However, symptomatic and physiologic improvement occurs in only a minority of patients, and neither smoking cessation nor immunosuppressive therapy is regularly associated with clinically significant benefit.BACKGROUND The clinical and physiologic features of respiratory bronchiolitis (RB)-interstitial lung disease (ILD) have been previously described; however, the natural history and outcome have not been systematically evaluated. The majority of published reports consider RB-ILD to be a nonprogressive ILD that clinically improves with smoking cessation and antiinflammatory treatment. In this study, we sought to determine the outcome of RB-ILD patients with and without smoking cessation and with and without corticosteroid therapy. METHODS Thirty-two RB-ILD cases confirmed by surgical lung biopsy were identified from a prospectively enrolled cohort of subjects with ILD. Initial and follow-up data on symptoms, physiology, treatment, and outcome were collected and analyzed. RESULTS Kaplan-Meier analysis revealed that at least 75% of RB-ILD patients survived > 7 years after diagnosis. Clinical improvement occurred in only 28% of cases, and physiologic improvement occurred in 10.5% of cases. One patient died of progressive ILD, and two patients died of non-small cell lung cancer. While physiologic improvement was limited to those who had ceased smoking, corticosteroids and/or other immunosuppressive therapy had little effect on symptoms or physiology. CONCLUSIONS This study shows that prolonged survival is common in RB-ILD. However, symptomatic and physiologic improvement occurs in only a minority of patients, and neither smoking cessation nor immunosuppressive therapy is regularly associated with clinically significant benefit.

Localized expression of tenascin in systemic sclerosis–associated pulmonary fibrosis and its regulation by insulin‐like growth factor binding protein 3

OBJECTIVE To determine the role of insulin-like growth factor binding protein 3 (IGFBP-3) in mediating the effects of transforming growth factor β (TGFβ) on tenascin-C (TN-C) production and to assess the levels of TN-C in vivo in patients with systemic sclerosis (SSc)-associated pulmonary fibrosis. METHODS Human primary lung fibroblasts were stimulated with TGFβ or IGFBP-3 in the presence or absence of specific small interfering RNAs and chemical inhibitors of the signaling cascade. TN-C levels in lung tissue specimens obtained from patients with SSc-associated pulmonary fibrosis were assessed using immunohistochemical analysis and were compared with the levels in specimens obtained from normal donors. TN-C levels were quantified in sera from normal donors and patients with SSc with or without pulmonary fibrosis, using an enzyme-linked immunosorbent assay. RESULTS IGFBP-3 mediated the induction of TN-C by TGFβ. Direct induction of TN-C by IGFBP-3 occurred in a p38 MAP kinase-dependent manner. TN-C levels were abundant in lung tissues from patients with SSc and were localized to subepithelial layers of the distal airways. No TN-C was detectable around the proximal airways. Patients with SSc-associated pulmonary fibrosis had significantly higher levels of circulating TN-C compared with SSc patients without pulmonary fibrosis. Longitudinal samples obtained from patients with SSc before and after the onset of pulmonary fibrosis showed increased levels of TN-C after the onset of pulmonary fibrosis. CONCLUSION IGFBP-3, which is overexpressed in fibrotic lungs, induces production of TN-C by subepithelial fibroblasts. The increased lung tissue levels of TN-C parallel the levels detected in the sera of SSc patients with pulmonary fibrosis, suggesting that TN-C may be a useful biomarker for SSc-related pulmonary fibrosis.

Role of Insulin-like Growth Factor Binding Protein-3 in Allergic Airway Remodeling

RATIONALE The hallmarks of allergic asthma are airway inflammation, obstruction, and remodeling. Airway remodeling may lead to irreversible airflow obstruction with increased morbidity and mortality. Despite advances in the treatment of asthma, the mechanisms underlying airway remodeling are still poorly understood. We reported that insulin-like growth factor (IGF) binding proteins (IGFBPs) contribute to extracellular matrix deposition in idiopathic pulmonary fibrosis; however, their contribution to airway remodeling in asthma has not been established. OBJECTIVES We hypothesized that IGFBP-3 is overexpressed in asthma and contributes to airway remodeling. METHODS We evaluated levels of IGFBP-3 in tissues and bronchoalveolar lavage fluid from patients with asthma at baseline and 48 hours after allergen challenge, in reparative epithelium in an in vitro wounding assay, and in conditioned media from cytokine- and growth factor-stimulated primary epithelial cells. MEASUREMENTS AND MAIN RESULTS IGFBP-3 levels and distribution were evaluated by Western blot, ELISA, and immunofluorescence. IGFBP-3 is increased in vivo in the airway epithelium of patients with asthma compared with normal control subjects. The concentration of IGFBP-3 is increased in the bronchoalveolar lavage fluid of patients with asthma after allergen challenge, its levels are increased in reparative epithelium in an in vitro wounding assay and in the conditioned medium of primary airway epithelial cell cultures stimulated with IGF-I. CONCLUSIONS Our results suggest that one mechanism of allergic airway remodeling is through the secretion of the profibrotic IGFBP-3 from IGF-I-stimulated airway epithelial cells during allergic inflammation.

Pathogenesis of Pulmonary Fibrosis in Systemic Sclerosis: Lessons from Interstitial Lung Disease

Interstitial lung disease is a frequent complication of systemic sclerosis and currently is the leading cause of death. Our ability to predict which individuals are at greatest risk of developing clinically significant, progressive interstitial lung disease remains inadequate. Identification of circulating autoantibodies and other biomarkers, as well as genetic polymorphisms and aberrant gene expression, all hold promise as diagnostic and prognostic tools, as well as therapeutic targets. Many practice patterns for the diagnosis and monitoring of connective tissue disease-associated interstitial lung disease are based upon published experience with idiopathic interstitial lung diseases. Although there are likely commonalities in the pathophysiologic mechanisms and clinical progression among all fibrosing lung diseases, a better understanding of features unique to systemic sclerosis-associated interstitial lung disease is essential to the development of more effective monitoring and treatment strategies.

Insulin-Like Growth Factor Binding Proteins-3 and -5: Central Mediators of Fibrosis and Promising New Therapeutic Targets

Fibrosis involves an orchestrated cascade of events including activation of fibroblasts, increased production and deposition of extracellular matrix components, and differentiation of fibroblasts into myofibroblasts. Epithelial-mesenchymal cross-talk plays an important role in this process, and current hypotheses of organ fibrosis liken it to an aberrant wound healing response in which epithelial-mesenchymal transition (EMT) and cellular senescence may also contribute to disease pathogenesis. The fibrotic response is associated with altered expression of growth factors and cytokines, including increased levels of transforming growth factor-β1 (TGF-β1) and the more recent observation that increased levels of several insulin-like growth factor binding proteins (IGFBPs) are associated with a number of fibrotic conditions. IGFBPs have been implicated in virtually every cell type and process associated with the fibrotic response, making the IGFBPs attractive targets for the development of novel anti-fibrotic therapies. In this review, the current state of knowledge regarding the classical IGFBP family in organ fibrosis will be summarized and the clinical implications considered.

Isolation and characterization of polyadenylation complexes assembled in vitro.

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.

Microbiome in lung explants of idiopathic pulmonary fibrosis: a case–control study in patients with end-stage fibrosis

The microbiome has been proposed to play a role in the progression of idiopathic pulmonary fibrosis (IPF) based on bronchoalveolar lavage analyses, but the microbiome of lung tissue in IPF has not been explored. In a case–control study of lung explants analysed by 16S rRNA gene sequencing, we could not reliably detect bacterial DNA in basilar tissue samples from patients with either chronic or acute exacerbations of IPF, in contrast to control candidate-donor lungs or cystic fibrosis explants. Thus, our data do not indicate microbiome alterations in regions of IPF lung with advanced fibrosis.