Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris.

Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously immortalized human keratinocyte cell line HaCaT or in primary keratinocytes. However, both cell systems have suboptimal features, with HaCaT cells exhibiting a large number of chromosomal aberrations and mutated p53 tumor suppressor, whereas primary keratinocytes are short-lived, heterogeneous and not susceptible to genetic modifications due to their restricted life-span. We have here tested the suitability of the commercially available human keratinocyte cell line hTert/KER-CT as a model system for research on epidermal cell adhesion and Pemphigus pathomechanisms. We here show that hTert cells exhibit a calcium dependent expression of desmosomal cadherins and are well suitable for typical assays used for studies on Pemphigus, such as sequential detergent extraction and Dispase-based dissociation assay. Treatment with Pemphigus auto-antibodies results in loss of monolayer integrity and altered localization of desmoglein-3, as well as loss of colocalization with flotillin-2. Our findings demonstrate that hTert cells are well suitable for studies on epidermal cell adhesion and Pemphigus pathomechanisms.

MicroRNA Monitoring in Human Alveolar Macrophages from Patients with Smoking-Related Lung Diseases: A Preliminary Study.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is commonly considered to be a potent driver of non-small cell lung cancer (NSCLC) development and related mortality. A growing body of evidence supports a role of the immune system, mainly played by alveolar macrophages (AMs), in key axes regulating the development of COPD or NSCLC phenotypes in response to harmful agents. MicroRNAs (miRNAs) are small non-coding RNAs that influence most biological processes and interfere with several regulatory pathways. The purpose of this study was to assess miRNA expression patterns in patients with COPD, NSCLC, and ever- or never-smoker controls to explore their involvement in smoking-related diseases. Bronchoalveolar lavage (BAL) specimens were collected from a prospective cohort of 43 sex-matched subjects to determine the expressions of hsa-miR-223-5p, 16-5p, 20a-5p, -17-5p, 34a-5p and 106a-5p by RT-PCR. In addition, a bioinformatic analysis of miRNA target genes linked to cancer was performed. Distinct and common miRNA expression levels were identified in each pathological group, suggesting their possible role as an index of NSCLC or COPD microenvironment. Moreover, we identified miRNA targets linked to carcinogenesis using in silico analysis. In conclusion, this study identified miRNA signatures in AMs, allowing us to understand the molecular mechanisms underlying smoking-related conditions and potentially providing new insights for diagnosis or pharmacological treatment.

The Role of MMPs in the Era of CFTR Modulators: An Additional Target for Cystic Fibrosis Patients?

Cystic fibrosis (CF) is a high-prevalence disease characterized by significant lung remodeling, responsible for high morbidity and mortality worldwide. The lung structural changes are partly due to proteolytic activity associated with inflammatory cells such as neutrophils and macrophages. Matrix metalloproteases (MMPs) are the major proteases involved in CF, and recent literature data focused on their potential role in the pathogenesis of the disease. In fact, an imbalance of proteases and antiproteases was observed in CF patients, resulting in dysfunction of protease activity and loss of lung homeostasis. Currently, many steps forward have been moved in the field of pharmacological treatment with the recent introduction of triple-combination therapy targeting the CFTR channel. Despite CFTR modulator therapy potentially being effective in up to 90% of patients with CF, there are still patients who are not eligible for the available therapies. Here, we introduce experimental drugs to provide updates on therapy evolution regarding a proportion of CF non-responder patients to current treatment, and we summarize the role of MMPs in pathogenesis and as future therapeutic targets of CF.

Exposure to low levels of hexavalent chromium: target doses and comparative effects on two human pulmonary cell lines.

Intracellular reduction of hexavalent chromium [Cr(VI)] is associated with the production of reactive oxygen species (ROS) and subsequent oxidative damage to different intracellular molecules like DNA, proteins and lipids is believed to contribute to the process of carcinogenesis. Aim of this study was to develop a model to establish a relationship between intracellular and macromolecule-bound chromium and some biomarkers of oxidative stress in two in vitro cell lines. Human lung adenocarcinoma (A549) and human bronchial epithelial (BEAS2B) cells were exposed for 3, 8 and 24 hours to relatively low doses (0.5--1--2 microM) of Cr(VI), i.e., to concentrations similar to what measured and reported by some authors in unexposed subjects and chromate workers. The results show that the differential cytotoxicity of Cr(VI) on the A549 and BEAS2B cell lines may be related both to their different polymorphism of Glutathione S-transferases genes and probably to their unlike permeability to Cr(VI). The glutathione decrease and the induction of HO-1 observed only in BEAS2B cells after Cr(VI) exposure strengthen the idea that glutathione S-transferases activity may accelerate the reduction of Cr(VI) to Cr(III) with the concomitant induction of oxidative stress. In conclusion, the determination of intracellular Cr in cellular models can be considered an important step in comparing in vitro and in vivo models on the basis of target doses and a promising approach to study the effects of pneumotoxic compounds.

A point mutation in the lariat branch point of intron 6 of NPC1 as the cause of abnormal pre-mRNA splicing in Niemann-Pick type C disease.

The lariat branch point sequence (BPS) is crucial for splicing pre-mRNA even if BPS mutations have infrequently been reported in human disease. In two siblings with Niemann-Pick type C (NPC) disease we identified two mutations of the NPC1 gene: i) one in exon 20 (c.2932C>T) (p.R978C) previously reported in NPC patients; ii) the other (c.882-28A>G) unreported, in the highly conserved adenosine of a putative lariat BPS of intron 6. Using RT-PCR we found that, besides the normally spliced mRNA, patients' fibroblasts contained minute amounts of an mRNA devoid of exon 7. The exon 6--exon 8 junction in this mRNA causes a frameshift and a premature stop codon, predicted to result in a truncated protein. To assess the effect of c.882-28A>G mutation we constructed two minigenes (wild type and mutant), spanning from intron 5 to intron 8, which were inserted into a pTarget vector and transfected in COS1 cells. The wild type minigene generated an mRNA of the expected size and sequence; the mutant minigene generated only an mRNA devoid of exon 7. This is the first example of a splicing defect due to a mutation in the lariat BPS in an intron of NPC1 found in NPC patients.

Prognostic role of clusterin in resected adenocarcinomas of the lung.

RATIONALE: Clusterin expression may change in various human malignancies, including lung cancer. Patients with resectable non-small cell lung cancer (NSCLC), including adenocarcinoma, have a poor prognosis, with a relapse rate of 30-50% within 5 years. Nuclear factor kB (Nf-kB) is an intracellular protein involved in the initiation and progression of several human cancers, including the lung. OBJECTIVES: We investigate the role of clusterin and Nf-kB expression in predicting the prognosis of patients with early-stage surgically resected adenocarcinoma of the lung. FINDINGS: The level of clusterin gradually decreased from well-differentiated to poorly differentiated adenocarcinomas. Clusterin expression was significantly higher in patients with low-grade adenocarcinoma, in early-stage disease and in women. Clusterin expression was inversely related to relapse and survival in both univariate and multivariate analyses. Finally, we observed an inverse correlation between Nf-kB and clusterin. CONCLUSIONS: Clusterin expression represents an independent prognostic factor in surgically resected lung adenocarcinoma and was proven to be a useful biomarker for fewer relapses and longer survival in patients in the early stage of disease. The inverse correlation between Nf-kB and clusterin expression confirm the previously reported role of clusterin as potent down regulator of Nf-kB.

Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke.

RATIONALE: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress. OBJECTIVES: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress. METHODS: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76-80 kD) and the 76-80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. CONCLUSIONS: We conclude that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts.