Identification of potential genes in endobronchial tuberculosis after bronchoscopic cryotherapy by transcriptome sequencing.

BACKGROUND: Bronchoscopic cryotherapy facilitates the treatment of endobronchial tuberculosis (EBTB) and helps suppress progressive bronchial stenosis. However, the molecular mechanism of bronchoscopic cryotherapy in EBTB patients has not been reported. METHODS: Transcriptome sequencing was performed to explore differentially expressed mRNAs (DEGs) in EBTB patients before and after bronchoscopic cryotherapy. Gene Ontology (GO) and KEGG analyses were carried out. Five genes (MKLN1, HIGD1A, PTGES, SKIL, and MCEMP1) were selected and validated using real-time qPCR (RT- qPCR). RESULTS: In transcriptome analysis, 448 DEGs with p < 0.05 and|logFC| > 1 were identified; of these, 171 and 277 DEGs were significantly up- and down-regulated after bronchoscopic cryotherapy, respectively. Results displayed 337 biological process (GO-BP), 48 cellular component (GO-CC) and 62 molecular function (GO-MF) terms and 20 KEGG pathways. RT- qPCR results were consistent with the sequencing data. CONCLUSION: These data provide informative evidence and recommendations for further scientific research on bronchoscopic cryotherapy for EBTB.

Altered Cell-Cycle Control, Inflammation, and Adhesion in High-Risk Persistent Bronchial Dysplasia.

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention.Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971-83. ©2018 AACR.

Congenital tracheobronchial stenosis.

Congenital tracheobronchial stenosis is a rare disease characterized by complete tracheal rings that can affect variable lengths of the tracheobronchial tree. It causes high levels of morbidity and mortality both due to the stenosis itself and to the high incidence of other associated congenital malformations. Successful management of this complex condition requires a highly individualized approach delivered by an experienced multidisciplinary team, which is best delivered within centralized units with the necessary diverse expertise. In such settings, surgical correction by slide tracheoplasty has become increasingly successful over the past 2 decades such that long-term survival now exceeds 88%, with normalization of quality of life scores for patients with non-syndrome-associated congenital tracheal stenosis. Careful assessment and planning of treatment strategies is of paramount importance for both successful management and the provision of patients and carers with accurate and realistic treatment counseling.

Gene expression profiling and pathway analysis of human bronchial epithelial cells exposed to airborne particulate matter collected from Saudi Arabia.

Epidemiological studies have established a positive correlation between human mortality and increased concentration of airborne particulate matters (PM). However, the mechanisms underlying PM related human diseases, as well as the molecules and pathways mediating the cellular response to PM, are not fully understood. This study aims to investigate the global gene expression changes in human cells exposed to PM(10) and to identify genes and pathways that may contribute to PM related adverse health effects. Human bronchial epithelial cells were exposed to PM(10) collected from Saudi Arabia for 1 or 4 days, and whole transcript expression was profiled using the GeneChip human gene 1.0 ST array. A total of 140 and 230 genes were identified that significantly changed more than 1.5 fold after PM(10) exposure for 1 or 4 days, respectively. Ingenuity Pathway Analysis revealed that different exposure durations triggered distinct pathways. Genes involved in NRF2-mediated response to oxidative stress were up-regulated after 1 day exposure. In contrast, cells exposed for 4 days exhibited significant changes in genes related to cholesterol and lipid synthesis pathways. These observed changes in cellular oxidative stress and lipid synthesis might contribute to PM related respiratory and cardiovascular disease.

Different risk factors between interstitial lung disease and airway disease in rheumatoid arthritis.

OBJECTIVE: To identify clinical and genetic risk factors for interstitial lung disease (ILD) or airway disease (AD) in patients with rheumatoid arthritis (RA) and to evaluate differences between the associations of these factors with ILD and AD. METHODS: We reviewed high-resolution computed tomography (HRCT) images and clinical data of 356 RA patients obtained at their first visit. The diagnosis of ILD and AD was based on abnormal HRCT findings. Multinomial logistic regression analysis and likelihood ratio tests were performed. RESULTS: High titers of rheumatoid factor are similarly associated with increased risks of ILD (relative risk ratio, 3.1; p = 0.02) and AD (relative risk ratio, 3.0; p = 0.02). High levels of anti-cyclic citrullinated peptide antibodies were associated strongly with AD (relative risk ratio, 3.8; p = 0.005) and less strongly with ILD (relative risk ratio, 2.7; p = 0.07). Age was the potent risk factor for ILD (relative risk ratio, 4.6; p = 0.003), while that for AD was advanced stage (relative risk ratio, 11.5; p < 0.0005). The carriage of HLA-DRB1*1502 had opposite influences on the two conditions: relative risk ratio = 4.02 for ILD (p = 0.013) and relative risk ratio = 0.15 for AD (p = 0.08). This difference was statistically significant (p = 0.0005). Associations of sex and smoking history with ILD disappeared in the multinomial logistic regression analysis. CONCLUSIONS: The differential associations of ILD and AD with various clinical and genetic factors suggest that ILD and AD have distinct etiologies in RA.

[Immunogenetic predisposition to chronic nonspecific inflammatory bowel disease].

Immunology has grown beyond the classic doctrine of immunity to infectious diseases, and gradually covered the problems of general physiology and pathology, genetics, embryology, transplantation, oncology and many other disciplines. A new direction has appeared--immunogenetics, which should help to answer questions the disposition and/or resistance to disease, as well as influence of environmental factors on implementation of predisposition to the development of pathology. Much attention is paid to investigation of HLA in IBD. These data indicate a significant polymorphism of major histocompatibility complex antigens in this disease in different countries. The aim of our study was to investigate the immunogenetic susceptibility and resistance to the development of ulcerative colitis (UC), and Crohn's disease (CD), the character of their flow, as well as the associated extraintestinal manifestations, in particular predisposition development of bronchial obstruction (BO) in patients with inflammatory bowel disease (IBD). A study of DNA frozen blood samples of 75 patients with IBD of both sexes has been conducted. The obtained results have been compared with the results of the study 1700 of samples of umbilical cord blood of newborns (apparently healthy children), born at 37-41 weeks' gestation in Moscow (control). The group of patients with UC revealed a positive association of HLA specificities-B*38, HLA-Cw*12 and HLA-DRV1*15, which can be regarded as potentially high risk of developing the disease. The presence of the specificity of HLA-DQV1*02 can be considered as a factor in resistance to the development of UC. High risk of developing Crohn's disease among residents of Moscow associated with groups of alleles of HLA B*41, HLA-B*56, HLA-Cw*05, HLA-Cw*08, HLA-DRV1*01, HLA-DRV1*11, HLA-DQV1*04, and the presence of specificity of HLA-DQV1*05 can be considered as a factor of resistance to the development of BC. High risk of developing BO in patients with IBD is associated with specificities HLA-DQB1*02, DQB1*03, DRB1*15.

The EvA study: aims and strategy.

The EvA study is a European Union-funded project under the Seventh Framework Programme (FP7), which aims at defining new markers for chronic obstructive pulmonary disease (COPD) and its subtypes. The acronym is derived from emphysema versus airway disease, indicating that the project targets these two main phenotypes of the disease. The EvA study is based on the concept that emphysema and airway disease are governed by different pathophysiological processes, are driven by different genes and have differential gene expression in the lung. To define these genes, patients and non-COPD controls are recruited for clinical examination, lung function analysis and computed tomography (CT) of the lung. CT scans are used to define the phenotypes based on lung density and airway wall thickness. This is followed by bronchoscopy in order to obtain samples from the airways and the alveoli. These tissue samples, along with blood samples, are then subjected to genome-wide expression and association analysis and markers linked to the phenotypes are identified. The population of the EvA study is different from other COPD study populations, since patients with current oral glucocorticoids, antibiotics and exacerbations or current smokers are excluded, such that the signals detected in the molecular analysis are due to the distinct inflammatory process of emphysema and airway disease in COPD.

Matrix metalloproteinase-1 polymorphism in Taiwanese patients with endobronchial tuberculosis.

Endobronchial tuberculosis (TB) often leads to some degree of tracheobronchial stenosis. Because matrix metalloproteinases (MMPs) play an essential role in tissue remodeling in the airways, we investigated the role of MMP-1 polymorphism in patients with endobronchial TB. One hundred and one cases of pulmonary TB in Taiwanese patients were genotyped for the 1G/2G polymorphism of MMP-1 promoter (-1607 bp). Bronchoscopic examination was performed to determine the presence of endobronchial involvement. Levels of MMP-1 in peripheral blood monocytes and in bronchial biopsies were also determined. 1G genotypes of MMP-1 polymorphism, containing at least one 1G allele, were associated with the presence of endobronchial TB. Using multivariate analysis, 1G genotypes and female gender were independent predictors of the development of endobronchial TB. Endobronchial TB patients with 1G genotypes had a 9.86-fold greater risk of developing tracheobronchial stenosis. IL-1beta increased levels of MMP-1 in peripheral blood monocytes of TB patients with 1G genotypes. MMP-1 activity was also present in the endobronchial TB granuloma from patients with 1G/1G genotype. 1G genotypes of MMP-1 polymorphism were associated with a greater risk of developing tracheobronchial stenosis through up-regulation of MMP-1 activity.

IL-21 receptor signaling is integral to the development of Th2 effector responses in vivo.

Interleukin 21 (IL-21) is a member of the common gamma-chain family of cytokines, which influence a broad spectrum of immunologic responses. A number of studies have examined the function of IL-21, but its specific role in Th1/Th2-cell differentiation and related effector responses remains to be clarified. Thus, we generated IL-21R-deficient mice and have investigated the role of IL-21R signaling using a series of in vivo experimentally induced disease models. We first addressed the role of IL-21R signaling in Th2 immune responses by examining allergic airway inflammation, and Nippostrongylus brasiliensis and Heligmosomoides polygyrus antihelminth responses. In each of these systems, IL-21R signaling played a clear role in the development of Th2 responses. Comparatively, IL-21R signaling was not required for the containment of Leishmania major infection or the development of experimental autoimmune myocarditis, indicative of competent Th1 and Th17 responses, respectively. Adoptive transfer of T cells and analysis of IL-21R+/+/IL-21R-/- chimera mice revealed that IL-21R-signaling was central to Th2-cell survival or migration to peripheral tissues. Overall, our data show IL-21 plays a crucial role in supporting polarized Th2 responses in vivo, while appearing superfluous for Th1 and Th17 responses.

Tracheobronchial calcification associated with Keutel syndrome.

Tracheobronchial cartilage calcification is an unusual radiologic finding in infants and children under 15 years old. Keutel syndrome is a rare, autosomal recessive disorder characterized by diffuse cartilage calcification, brachytelephalangia, pulmonary stenosis and midfacial hypoplasia. We report two children in whom abnormal tracheobronchial calcification was associated with Keutel syndrome. Keutel syndrome should be considered in the differential diagnosis of children with tracheobronchial calcification.

Underlying chronic granulomatous disease in a patient with bronchocentric granulomatosis.

We present a case of bronchocentric granulomatosis in a woman with no history of asthma who was colonised with Aspergillusfumigatus. A family history of chronic granulomatous disease prompted further testing that demonstrated severely depressed neutrophil oxidant production and gp91(phox) deficiency compatible with the X linked carrier state of chronic granulomatous disease. Only one report of the association of these two rare diseases has previously appeared in the literature. We postulate that an ineffective immune response led to the prolonged colonisation of Afumigatus resulting in a hypersensitivity reaction that was manifest clinically as bronchocentric granulomatosis.

Molecular follow-up of preneoplastic lesions in bronchial epithelium of former Chernobyl clean-up workers.

Ionizing radiation is a potent lung carcinogen, but the precise molecular damage associated with it is still unknown. In this study we investigated cancer-related molecular abnormalities including K-ras (codon 12) mutation, p16(INK4A) promoter hypermethylation and microsatellite alterations at seven chromosomal regions in successive biopsies obtained from former Chernobyl cleanup workers in comparison with smokers and nonsmokers who have never had radiation exposure. Our results indicate that prolonged persistence of inhaled radioactive particles is associated with appearance of allelic loss at 3p12, 3p14.2 (FHIT), 3p21, 3p22-24 (hMLH1) and 9p21 (p16INK4A) in bronchial epithelium of former Chernobyl clean-up workers. The prevalence of 3p14.2 allelic loss was associated with decreased expression of the FHIT mRNA in their bronchial epithelium in comparison with control group of smokers. During several years of our monitoring samples of epithelium were collected from the same area of bronchial tree. In epithelium exposed to carcinogens (tobacco smoke and/or radioactivity) the total number of molecular abnormalities was significantly higher in dysplasia and in morphologically normal foci progressed later to dysplasia than in these samples which never showed evidence of such progression. Our findings indicate that extensive cancer-related molecular abnormalities sequentially occur in radiation damaged bronchial epithelium of former Chernobyl clean-up workers.

Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations.

Loss of heterozygosity (LOH) involving chromosomes 3p, 5q, 9p, or 17p and aberrant expression or mutation of p53 are reported previously in selected bronchial dysplasias and squamous cell cancers (SCCs). Yet, comprehensive analyses of LOH patterns at these chromosomal sites and of p53 alterations are not reported for histologically normal bronchial epithelium, high-grade bronchial dysplasia, and SCC present in the same pulmonary resections. Whether concordant or discordant genetic changes are detected in these bronchial tissues, especially when multiple high-grade dysplastic bronchial lesions are present, was studied. Genomic DNA was microdissected from eight pulmonary SCCs and high-grade dysplastic lesions that were associated with SCC. In four cases, two independent high-grade dysplastic bronchial lesions were identified. When available, histologically normal bronchial epithelium was microdissected. Germ-line genomic DNA was isolated from normal lymph nodes. LOH was assessed for 15 microsatellite markers on chromosomes 3p, 5q, 9p, or 17p, sites frequently deleted in lung cancers. Immunohistochemical p53 expression was studied and correlated with p53 DNA sequence analyses. Progressive LOH for these markers was found when SCCs were compared with high-grade dysplasia and histologically normal bronchial epithelium present in the same resections. Histologically normal bronchial specimens had LOH in up to 27% of informative markers. High-grade dysplastic lesions exhibited LOH for 18-45% and SCC had LOH for 18-73% of the markers. Common regions of LOH were found in some dysplasias compared with SCCs. In other dysplasias, discordance was found relative to SCCs, especially for p53 mutations. In cases with a single or second high-grade dysplasia associated with SCC, heterogeneity in LOH markers was detected. These concordant and discordant changes were consistent with convergent and divergent clonal selection pathways in pulmonary squamous cell carcinogenesis. Some histologically normal bronchial epithelial tissues had genetic changes more similar to those in the SCCs than in dysplastic lesions. DNA loss or mutations accumulate in SCC, but discordant genetic changes can exist in the same carcinogen-exposed bronchial tissues. These findings have implications for lung cancer prevention trials.

Cyclin proteolysis as a retinoid cancer prevention mechanism.

The retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and prevention. Their biological effects are mediated through ligand-dependent interactions with retinoid receptors that associate with specific co-regulators. A better understanding of retinoid chemopreventive mechanisms is needed. Our prior work revealed that all-trans-retinoic acid (RA) prevented tobacco-specific carcinogenic transformation of cultured human bronchial epithelial cells. RA signaled G1 arrest that permitted repair of genomic DNA damage caused by these carcinogens. RA triggered G1 arrest at least partly through proteasome-dependent degradation of cyclin D1. Proteasomal inhibitors blocked RA-mediated cyclin D1 degradation. To confirm that a specific proteolysis pathway was induced by RA-treatment, a degradation assay was established using in vitro translated cyclin D1 and cellular extracts from RA-treated or untreated human bronchial epithelial cells. Incubation of RA-treated but not the control cellular extracts with in vitro translated cyclin D1 led to cyclin degradation. This degradation depended on the PEST domain of cyclin D1, implicating ubiquitination in this retinoid degradation. Retinoid receptor selective agonists demonstrated that retinoic acid receptor (RAR)beta and retinoid X receptor (RXR) but not RARalpha- or RARgamma-dependent pathways signaled this cyclin degradation. Findings were extended to the NT2/D1 human embryonal carcinoma differentiation model where a similar pathway was activated by RA-treatment. To determine whether G1 cyclins were involved directly in bronchial preneoplasia, immunohistochemical expression profiles for cyclins D1 and E were examined. Aberrant expression of these cyclins was frequent in bronchial preneoplasia. Taken together, these findings indicate that ubiquitin-dependent proteolysis of G1 cyclins is a retinoid chemoprevention mechanism. Whether the retinoids represent the optimal agents to activate this pathway is the subject of ongoing work. These findings provide a rationale for combining the retinoids in chemoprevention trials with other agents that do not activate this proteolysis pathway. What is now known about the retinoids as cancer prevention agents will be reviewed. Emphasis is placed on retinoid effects on cell cycle progression at G1.

Overexpression of cyclins D1 and E is frequent in bronchial preneoplasia and precedes squamous cell carcinoma development.

Increased protein expression of the G1 cyclins D1 and E is reported in invasive non-small cell lung carcinoma. However, during transformation of the bronchial epithelium, overexpression of these species occurs, and their relationship to aberrant expression of p53 and retinoblastoma (Rb) has not been described previously. To determine the expression of these cell cycle regulators during the development of invasive squamous cell carcinoma (SCC) of the lung, the immunohistochemical expression patterns in normal bronchial epithelium (n = 36), squamous metaplasia (SM; n = 28), and epithelial atypia (n = 34) were compared with that in low-grade dysplasia (LGD; n = 17), high-grade bronchial dysplasia (HGD; n = 30), and SCC (n = 36). Monoclonal anti-p53 Pab1801, polyclonal anti-cyclin D1 DCS6, monoclonal anti-cyclin E HE12, and monoclonal anti-Rb OP-66 antibodies were used. Cyclin D1 was not expressed in normal bronchial epithelium but was detected in 7% of SMs, 15% of atypias; 18% of LGDs, 47% of HGDs, and 42% of SCCs. Cyclin E was not detected in normal epithelium (n = 24), SM (n = 16), or LGD (n = 12), but it was found in 9% of atypias (2 of 22), 33% of HGDs (7 of 21), and 54% of SCCs (13 of 24). p53 was not expressed in normal epithelium, SM, and LGD, but it was overexpressed in 6% of atypias, 53% of HGDs, and 61% of SCCs. Abnormal Rb expression was found only in 2 of 36 cases of SCC. A total of 91% of HGDs and 92% of SCCs exhibited overexpression of at least one of the p53, cyclin D1, or cyclin E species. However, no link was observed between overexpression of p53 and the overexpressed G1 cyclins in preneoplastic lesions. Overexpression of cyclin D1, cyclin E, and p53 occurs frequently and independently in pulmonary SCC and is detected in lesions before the development of invasive carcinoma. In contrast, altered Rb expression is a late and infrequent event in squamous cell carcinogenesis.