Functional Wnt signaling is increased in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/β-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/β-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/β-catenin pathway in IPF.

Methodology/Principal Findings

The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3β, β-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, β-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, β-catenin, and Gsk-3β expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3β, phospho-Lrp6, and β-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/β-catenin signaling in IPF compared with controls. Functional in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis.

Conclusions/Significance

Our study demonstrates that the Wnt/β-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/β-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF.     

Probe selection algorithms with applications in the analysis of microbial communities

We propose two efficient heuristics for minimizing the number of oligonucleotide probes needed for analyzing populations of ribosomal RNA gene (rDNA) clones by hybridization experiments on DNA microarrays. Such analyses have applications in the study of microbial communities. Unlike in the classical SBH (sequencing by hybridization) procedure, where multiple probes are on a DNA chip, in our applications we perform a series of experiments, each one consisting of applying a single probe to a DNA microarray containing a large sample of rDNA sequences from the studied population. The overall cost of the analysis is thus roughly proportional to the number of experiments, underscoring the need for minimizing the number of probes. Our algorithms are based on two well-known optimization techniques, i.e. simulated annealing and Lagrangian relaxation, and our preliminary tests demonstrate that both algorithms are able to find satisfactory probe sets for real rDNA data.     

Expression of simian immunodeficiency virus nef in immune cells of transgenic mice leads to a severe AIDS-like disease

In order to study the functions of simian immunodeficiency virus (SIV) Nef in vivo in a small-animal model, we constructed transgenic (Tg) mice expressing the SIV(mac)239 nef gene in the natural target cells of the virus under the control of the human CD4 gene promoter (CD4C). These CD4C/SHIV-nef(SIV) Tg mice develop a severe AIDS-like disease, with manifestations including premature death, failure to thrive or weight loss, wasting, thymic atrophy, an especially low number of peripheral CD8+ T cells as well as a low number of peripheral CD4+ T cells, diarrhea, splenomegaly, and kidney (interstitial nephritis, segmental glomerulosclerosis), lung (lymphocytic interstitial pneumonitis), and heart disease. In addition, these Tg mice fail to mount a class-switched antibody response after immunization with ovalbumin, they produce anti-DNA autoantibodies, and some of them develop Pneumocystis carinii lung infections. All these results suggest a generalized Nef-induced immunodeficiency. The low numbers of peripheral CD8+ and CD4+ T cells are likely to reflect a thymic defect and may be similar to the DiGeorge-like "thymic defect" immunophenotype described for a subgroup of human immunodeficiency virus type 1-infected children. Therefore, it appears that SIV Nef alone expressed in mice, in appropriate cell types and at sufficient levels, can elicit many of the phenotypes of simian and human AIDS. These Tg mice should be instrumental in studying the pathogenesis of SIV Nef-induced phenotypes.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis

Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.     

Morningness–eveningness and affective temperaments assessed by the Temperament Evaluation of Memphis,Pisa and San Diego-Autoquestionnaire (TEMPS-A)

Chronotype is a stable trait presenting one’s circardian preference. Since chronotype disturbances are common in patients with affective disorders, our aim is to evaluate chronotypes related to affective temperaments, measured with the temperament evaluation of Memphis, Pisa and San Diego-Autoquestionnaire (TEMPS-A). The study included 618 subjects (151 men and 467 women) within the framework of web-based design. They all fulfilled a questionnaire, consisting of the Composite Scale of Morningness (CSM), Sleep Wake Pattern Assessment Questionnaire (SWPAQ), and the TEMPS-A scale. Multiple regression models revealed that after controlling for age and gender: irritable and cyclothymic temperaments were negatively associated with total CSM score, CSM morning affect and circadian preference components, Sleepability (S), Vigilance (V), Wakeability (W) and positively with Morningness (M) and Eveningness (E) subscales of SWPAQ; anxious temperament was negatively associated with total CSM scores, CSM morning affect and with S, V, W subscales of SWPAQ; depressive temperament was negatively associated with Falling asleep, S, V, W subscales of SWPAQ; hyperthymic temperament was positively associated with CSM morning affect and V, W and negatively with M subscales of SWPAQ. The results show distinctiveness of the associations between hyperthymic temperament and circadian preferences, compared to all other TEMPS-A temperaments (depressive, cyclothymic, irritable and anxious). In the CMS scale, only hyperthymic temperament was related to morning affect. In the SWPAQ scale, hyperthymic temperament was the only one associated with earlier morningness (earlier wake up time preference), increased parameters of vigor – wakeability, vigilance, and also the only one not associated with decreased plasticity of circadian rhythm (sleepability and falling asleep). Results also point to some similarities between cyclothymic and irritable temperaments in some aspects of the chronotype.     

Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.