Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells

<p>Abstract</p> <p>Background</p> <p>Alveolar epithelial cells are known as progenitor cells for the restoration from the damage in the lung. Calcitonin gene-related peptide (CGRP) has been reported to play an important role in the proliferation of various types of epithelial and endothelial cells. We investigated the effects of CGRP on the proliferation of alveolar epithelial cells <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>A549 cells were cultured in Dulbecco Modified Eagle Medium with 5% fatal bovin serum for 24 hours, then CGRP was added <it>in vitro</it>. The proliferation of DNA synthesis was measured using 5-bromo-2-deoxyuridine, an analog of thymidine, by enzyme-linked immunosorbent assay.</p> <p>As one intracellular response to CGRP, we examined activation of p44/42- extracellular signal-regulated kinase (ERK) pathway by adding CGRP, using western blotting method.</p> <p>Recombinant adenovirus encoding nuclear-targeted-human β-CGRP (rhCGRP) was administered into Male Wister rat (n = 5, 10 weeks old) lungs by intratracheal instillation <it>in vivo</it>. 7 days after the administration of CGRP, rat lungs were harvested and histological findings and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) were evaluated to examine cell proliferation.</p> <p>Results</p> <p><it>In vitro </it>study, CGRP increased the proliferation of A549 cells in a dose and time dependent manner. CGRP8-37 (inhibitor of CGRP receptor) decreased CGRP induced proliferation of DNA synthesis. Phosphorylation of ERK pathway was observed within 15 minutes and peaked in one hour. U0126 (inhibitor of ERK pathway) decreased CGRP induced proliferation of DNA synthesis.<it>In vivo </it>study, histological examination of the lung indicated proliferation of alveolar epithelial cells in the rhCGRP-treated group and the nuclei of alveolar epithelial cells were positive for PCNA immunostaining.</p> <p>Conclusion</p> <p>In this study, we conclude that CGRP stimulates proliferation of human alveolar epithelial cells <it>in vivo </it>and <it>in vitro</it>.</p

Anti-cytokine therapy in fibrosing alveolitis: where are we now?

Idiopathic pulmonary fibrosis (IPF) is a condition that has a poor prognosis, with a median survival of 4-5 years irrespective of treatment. Ziesche et al (N Engl J Med 1999, 341: 1264-1269) describe an open randomised trial of 18 patients with IPF, unresponsive to corticosteroid treatment at high dose. Nine patients were treated with continued corticosteroid and nine with prednisolone plus interferon-γ 1b (IFN-γ). Significant benefits in physiological parameters are reported in the IFN-γ-treated group. An analysis of lung tissue by reverse-transcriptase-mediated polymerase chain reaction showed corresponding decreases in the transcription of transforming growth factor-β1 and connective tissue growth factor. This is the first report of treatment showing efficacy in this disease, albeit in a very preliminary study, but the data should be viewed with caution. This study is discussed in the context of other published studies of treatment for IPF and the scientific rationale on which it was based

Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

BACKGROUND: Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. METHODS: Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. RESULTS: Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. CONCLUSION: The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers

Bronchoalveolar lavage fluid from preterm infants with chorioamnionitis inhibits alveolar epithelial repair

<p>Abstract</p> <p>Background</p> <p>Preterm infants are highly susceptible to lung injury. While both chorioamnionitis and antenatal steroids induce lung maturation, chorioamnionitis is also associated with adverse lung development. We investigated the ability of bronchoalveolar lavage fluid (BALF) from ventilated preterm infants to restore alveolar epithelial integrity after injury <it>in vitro</it>, depending on whether or not they were exposed to chorioamnionitis or antenatal steroids. For this purpose, a translational model for alveolar epithelial repair was developed and characterised.</p> <p>Methods</p> <p>BALF was added to mechanically wounded monolayers of A549 cells. Wound closure was quantified over time and compared between preterm infants (gestational age < 32 wks) exposed or not exposed to chorioamnionitis and antenatal steroids (≥ 1 dose). Furthermore, keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) were quantified in BALF, and their ability to induce alveolar epithelial repair was evaluated in the model.</p> <p>Results</p> <p>On day 0/1, BALF from infants exposed to antenatal steroids significantly increased epithelial repair (40.3 ± 35.5 vs. -6.3 ± 75.0% above control/mg protein), while chorioamnionitis decreased wound-healing capacity of BALF (-2.9 ± 87.1 vs. 40.2 ± 36.9% above control/mg protein). BALF from patients with chorioamnionitis contained less KGF (11 (0-27) vs. 0 (0-4) pg/ml) and less detectable VEGF (66 vs. 95%) on day 0. BALF levels of VEGF and KGF correlated with its ability to induce wound repair. Moreover, KGF stimulated epithelial repair dose-dependently, although the low levels in BALF suggest KGF is not a major modulator of BALF-induced wound repair. VEGF also stimulated alveolar epithelial repair, an effect that was blocked by addition of soluble VEGF receptor-1 (sVEGFr1/Flt-1). However, BALF-induced wound repair was not significantly affected by addition of sVEGFr1.</p> <p>Conclusion</p> <p>Antenatal steroids improve the ability of BALF derived from preterm infants to stimulate alveolar epithelial repair <it>in vitro</it>. Conversely, chorioamnionitis is associated with decreased wound-healing capacity of BALF. A definite role for KGF and VEGF in either process could not be established. Decreased ability to induce alveolar epithelial repair after injury may contribute to the association between chorioamnionitis and adverse lung development in mechanically ventilated preterm infants.</p

Simian virus 40 vectors for pulmonary gene therapy

<p>Abstract</p> <p>Background</p> <p>Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy.</p> <p>Methods</p> <p>Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/<it>luc) </it>were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by <it>in vivo </it>light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector.</p> <p>Results</p> <p>Luc expression measured by <it>in vivo </it>light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response.</p> <p>Conclusion</p> <p>In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of <it>in vivo </it>transduction of alveolar type II cells and may thus become a future therapeutic tool.</p

Risk factors associated with fatal pulmonary hemorrhage in locally advanced non-small cell lung cancer treated with chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to identify the risk factors associated with fatal pulmonary hemorrhage (PH) in patients with locally advanced non-small cell lung cancer (NSCLC), treated with chemoradiotherapy.</p> <p>Methods</p> <p>The medical records of 583 patients with locally advanced NSCLC, who were treated with chemoradiotherapy between July 1992 and December 2009 were reviewed. Fatal PH was defined as PH leading to death within 24 h of its onset. Tumor cavitation size was defined by the cavitation diameter/tumor diameter ratio and was classified as minimum (< 0.25), minor (≥ 0.25, but < 0.5), and major (≥ 0.5).</p> <p>Results</p> <p>Of the 583 patients, 2.1% suffered a fatal PH. The numbers of patients with minimum, minor, and major cavitations were 13, 11, and 14, respectively. Among the 38 patients with tumor cavitation, all 3 patients who developed fatal PH had major cavitations. On multivariate analysis, the presence of baseline major cavitation (odds ratio, 17.878), and a squamous cell histology (odds ratio, 5.491) proved to be independent significant risk factors for fatal PH. Interestingly, all patients with fatal PH and baseline major cavitation were found to have tumors with squamous cell histology, and the occurrence of fatal PH in patients having both risk factors was 33.3%.</p> <p>Conclusions</p> <p>Patients at high risk of fatal PH could be identified using a combination of independent risk factors.</p

Risk factors for ischaemic heart disease in a Cretan rural population: a twelve year follow-up study

<p>Abstract</p> <p>Background</p> <p>Crete has been of great epidemiological interest ever since the publication of the Seven Countries Study. In 1988 a well-defined area of rural Crete was studied, with only scarce signs of coronary heart disease (CHD) despite the unfavorable risk profile. The same population was re-examined twelve years later aiming to describe the trends of CHD risk factors over time and discuss some key points on the natural course of coronary heart disease in a rural population of Crete.</p> <p>Methods and Results</p> <p>We re-examined 200 subjects (80.7% of those still living in the area, 62.4 ± 17.0 years old). The prevalence of risk factors for CHD was high with 65.9% of men and 65.1% of women being hypertensive, 14.3% of men and 16.5% of women being diabetic, 44% of men being active smokers and more than 40% of both sexes having hyperlipidaemia. Accordingly, 77.5% of the population had a calculated Framingham Risk Score (FRS) ≥ 15%, significantly higher compared to baseline (p < 0.001). The overall occurrence rate for CHD events was calculated at 7.1 per 1000 person-years (95% confidence interval: 6.8–7.3).</p> <p>Conclusion</p> <p>The study confirms the unfavorable risk factor profile of a well defined rural population in Crete. Its actual effect on the observed incidence of coronary events in Cretans remains yet to be defined.</p

Prediction of HIV-1 virus-host protein interactions using virus and host sequence motifs

<p>Abstract</p> <p>Background</p> <p>Host protein-protein interaction networks are altered by invading virus proteins, which create new interactions, and modify or destroy others. The resulting network topology favors excessive amounts of virus production in a stressed host cell network. Short linear peptide motifs common to both virus and host provide the basis for host network modification.</p> <p>Methods</p> <p>We focused our host-pathogen study on the binding and competing interactions of HIV-1 and human proteins. We showed that peptide motifs conserved across 70% of HIV-1 subtype B and C samples occurred in similar positions on HIV-1 proteins, and we documented protein domains that interact with these conserved motifs. We predicted which human proteins may be targeted by HIV-1 by taking pairs of human proteins that may interact via a motif conserved in HIV-1 and the corresponding interacting protein domain.</p> <p>Results</p> <p>Our predictions were enriched with host proteins known to interact with HIV-1 proteins ENV, NEF, and TAT (p-value < 4.26E-21). Cellular pathways statistically enriched for our predictions include the T cell receptor signaling, natural killer cell mediated cytotoxicity, cell cycle, and apoptosis pathways. Gene Ontology molecular function level 5 categories enriched with both predicted and confirmed HIV-1 targeted proteins included categories associated with phosphorylation events and adenyl ribonucleotide binding.</p> <p>Conclusion</p> <p>A list of host proteins highly enriched with those targeted by HIV-1 proteins can be obtained by searching for host protein motifs along virus protein sequences. The resulting set of host proteins predicted to be targeted by virus proteins will become more accurate with better annotations of motifs and domains. Nevertheless, our study validates the role of linear binding motifs shared by virus and host proteins as an important part of the crosstalk between virus and host.</p

A Novel Solid-Phase Site-Specific PEGylation Enhances the In Vitro and In Vivo Biostabilty of Recombinant Human Keratinocyte Growth Factor 1

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1

The oxytocin analogue carbetocin prevents emotional impairment and stress-induced reinstatement of opioid-seeking in morphine-abstinent mice.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20-100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse