Whole exome sequencing identifies a rare variant in DAAM2 as a potential candidate in idiopathic pulmonary ossification.

BACKGROUND: Diffuse pulmonary ossification (DPO) is a rare disease characterized by bone tissue formation in the lung. DPO can be classified into idiopathic pulmonary ossification (IPO) and secondary pulmonary ossification. Cases with no identified etiology are classified as IPO. Variants of dishevelled associated activator of morphogenesis 2 (DAAM2) have been reported to be involved in the bone-resorption of osteoclasts. METHODS: Whole exome sequencing (WES) was used on samples from a patient with IPO and his healthy parents. The effects of all variants were determined using functional predictors (PolyPhen-2, SIFT, FATHMM and MutationTaster); variants existing only in the patient were further screened compared with his healthy parents. RESULTS: Forty deleterious variants, including 25 single nucleotide variants (SNVs) and 15 insertions and deletions (indels), were identified by WES. Finally, DAAM2 (c.G2960T:p.R987L) was screened by pathway analysis. CONCLUSIONS: We identified a novel variant of DAAM2 (c.G2960T:p.R987L) that might participate in the disease process of IPO.

Peripheral blood CD4+ T cell populations by CD25 and Foxp3 expression as a potential biomarker: reflecting inflammatory activity in chronic obstructive pulmonary disease.

Background: The temporally dynamic changes of CD25 and Foxp3 expression in CD4+ T cells are initiated by T cell receptor (TCR) signals strength or frequency. There is a deficiency of peripheral markers for assessing COPD activity, and the current study was conducted to explore whether peripheral CD4+ T cell populations based on CD25 and Foxp3 expression could serve as an indicator for COPD inflammatory activity. Methods: The distribution and phenotypic characteristics of CD4+CD25±Foxp3± T cells from peripheral blood in different populations were determined by flow cytometry. The model for the differentiation of CD4+ T cells populations by CD25 and Foxp3 expression was explored in vitro. Results: The frequencies of peripheral CD4+CD25+Foxp3- T cells and CD4+CD25+Foxp3+ T cells were increased in AECOPD patients, whereas the frequency of CD4+CD25-Foxp3+ T cells was increased in SCOPD patients without receiving systemic treatment. Phenotypic analysis revealed that CD4+CD25+Foxp3- T cells, CD4+CD25+Foxp3+ T cells and CD4+CD25-Foxp3+ T cells had received antigenic stimulation and resembled central memory or effector memory T cells. The differentiation of CD4+ T cells populations by CD25 and Foxp3 expression was dictated by TCR signals. The paired study indicated that the frequencies of CD4+CD25+Foxp3- T cells, CD4+CD25+Foxp3+ T cells and CD4+CD25- Foxp3+ T cells were decreased while the frequency of CD4+CD25-Foxp3- T cells were increased in the same patients from AECOPD to convalescence. Conclusions: Collectively, we propose that the dynamic changes of CD4+ T cell populations by CD25 and Foxp3 expression could function as potential biomarkers for reflecting inflammatory activity in COPD.

Generation and Immune Regulation of CD4+CD25-Foxp3+ T Cells in Chronic Obstructive Pulmonary Disease.

The imbalance of CD4+Foxp3+ T cell subsets is reportedly involved in abnormal inflammatory immune responses in patients with chronic obstructive pulmonary disease (COPD). However, the possible role of CD4+CD25-Foxp3+ T cells in immune regulation in COPD remains to be investigated. In the current study, distribution and phenotypic characteristics of CD4+CD25-Foxp3+ T cells from peripheral blood were determined by flow cytometry; the origin, immune function and ultimate fate of CD4+CD25-Foxp3+ T cells were further explored in vitro. It was observed that circulating CD4+CD25-Foxp3+ T cells were significantly increased in stable COPD patients (SCOPD) and resembled central memory or effector memory T cells. Compared with peripheral CD4+CD25+Foxp3+ T cells, peripheral CD4+CD25-Foxp3+ T cells showed a lower expression of Foxp3, CTLA-4, HELIOS, and TIGIT, but a higher expression of CD127 and KI-67, suggesting that CD4+CD25-Foxp3+ T cells lost the expression of Tregs-associated molecules following the reduction in CD25. Unexpectedly, our study found that transforming growth factor-ß1 (TGFß1) decreased CD25 expression and played a critical role in the generation of CD4+CD25-Foxp3+ T cells from CD4+CD25+Foxp3+ T cells. Phenotypic analysis further revealed that both inducible and peripheral CD4+CD25-Foxp3+ T cells exhibited the features of activated conventional T cells. Importantly, memory CD4+CD25-Foxp3+ T cells facilitated the proliferation and differentiation of naïve CD4+ T cells into Th17 cells in the presence of IL-1ß, IL-6, IL-23, and TGFß1. Finally, a fraction of CD4+CD25-Foxp3+ T cells, exhibiting instability and plasticity, were converted to Th17 cells when subjected to Th17 cell-polarizing condition. Taken together, we propose that TGFß1 is responsible for the generation of CD4+CD25-Foxp3+ T cells, and these cells functionally exert an auxiliary effect on Th17 cells generation and might perpetuate chronic inflammation in COPD.

BAMBI regulates macrophages inducing the differentiation of Treg through the TGF-ß pathway in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by continuous flow limitation and the immune system including macrophages and regulatory T lymphocytes (Tregs) is involved in COPD pathogenesis. In our previous study, we investigated that TGF-ß/BAMBI pathway was associated with COPD by regulating the balance of Th17/Treg. However, the role of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a pseudoreceptor of TGF-ß signalling pathway, in regulating the immune system of COPD patients has not been fully studied. Hence, we speculate that the pseudoreceptor BAMBI may play roles in the regulation of M2 macrophages to induce the differentiation of CD4+ naïve T cells into Tregs and influence the immune response in COPD. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy nonsmokers (n = 12), healthy smokers (n = 10) and COPD patients (n = 20). Naïve CD4+ T cells and monocytes-induced macrophages were used for coculture assays. The phenotypic characteristics of macrophages and Tregs were determined by flow cytometry. The expression levels of BAMBI and the TGF-ß/Smad pathway members in M2 macrophages were measured by a Western blot analysis. The monocyte-derived macrophages were stimulated with cigarette smoke extract (CSE, concentration of 0.02%) to simulate the smoking process in humans. pCMV-BAMBI was transfected into monocyte-derived M2 macrophages for subsequent co-culture assays and signalling pathway analysis. RESULTS: Our results showed that M2 macrophages could induce the differentiation of Tregs through the TGF-ß/Smad signalling pathway. In addition, monocyte-derived macrophages from COPD patients highly expressed BAMBI, and had a low capacity to induce Tregs differentiation. The expression of BAMBI and the forced expiratory volume in 1 second (FEV1%) were negatively correlated in COPD. Furthermore, overexpression of BAMBI promoted the conversion of M2 macrophages to M1 macrophages via the TGF-ß/Smad pathway. CONCLUSIONS: We demonstrated that BAMBI could promote the polarization process of M2 macrophages to M1 macrophages via the TGF-ß/Smad signalling pathway and that overexpression of BAMBI could decrease the ability of M2 macrophages to induce Treg differentiation. These findings may provide a potential mechanism by which blocking BAMBI could improve immune function to regulate COPD inflammatory conditions.

Neoadjuvant chemoradiotherapy could improve survival outcomes for esophageal carcinoma: a meta-analysis.

BACKGROUND: The effectiveness of neoadjuvant chemoradiotherapy in patients with resectable esophageal carcinoma remains controversial. AIMS: The purpose of this study was to assess the effect of neoadjuvant chemoradiotherapy on operable esophageal carcinoma. METHODS: We searched PubMed, EMBASE and Web of Science and identified all randomized controlled trials published up until July 2011 that directly compared chemoradiotherapy followed by surgery with surgery alone. The risk ratio (RR) with its corresponding 95 % confidence interval (CI) was the principal measure of effects. RESULTS: Twelve randomized controlled trials that met our inclusion criteria were identified. Chemoradiotherapy followed by surgery was associated with significantly improved 1-year (RR = 0.86, 95 % CI = 0.74-0.98, P = 0.03), 3-year (RR = 0.82, 95 % CI = 0.73-0.92, P = 0.0007) and 5-year (RR = 0.83, 95 % CI = 0.72-0.96, P = 0.01) survival times compared with surgery alone. Subgroup analysis suggested that this benefit was associated with concurrent chemoradiotherapy but not sequential chemoradiotherapy. Neoadjuvant chemoradiotherapy could improve 3- and 5-year survival outcomes for squamous cell carcinoma but not those for adenocarcinoma. Postoperative morbidity (RR = 0.97, 95 % CI = 0.86-1.09, P = 0.56) and mortality (RR = 1.56, 95 % CI = 0.97-2.50, P = 0.07) did not increase in patients treated by chemoradiotherapy. CONCLUSIONS: Our findings revealed that compared with surgery alone, neoadjuvant chemoradiotherapy was associated with improved 1-, 3- and 5-year survival times, but not associated with increased postoperative morbidity and mortality in patients with esophageal carcinoma.

Effects of RNAi-mediated matrix metalloproteinase-2 gene silencing on the invasiveness and adhesion of esophageal carcinoma cells, KYSE150.

BACKGROUND: Esophageal carcinoma is one of the main malignancies in China. Previous studies indicated that matrix metalloproteinases (MMPs) play important roles in the process of tumor invasion and metastasis in several types of solid tumors. Among all of the MMPs, MMP-2 is one of the MMPs closely associated with tumor invasion. In this study, we suppressed MMP-2 expression with RNA interference and then observed inhibitory effects on the invasion and migration of the esophageal carcinoma cell line KYSE150. METHODS: Three target sequences were selected and siRNA against MMP-2 mRNA were synthesized. After being transfected by the transfection complexes, the MMP-2 expression of KYSE150 cells, which overexpresses MMP-2, were examined by Western blot analysis and real-time polymerase chain reaction (PCR). Cell migration and invasion were measured with migration assay and Boyden chamber assays, respectively. RESULTS: RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the esophageal carcinoma cell line KYSE150. MMP-2 knockdown inhibited the invasion and migration of esophageal carcinoma cell line KYSE150. CONCLUSIONS: These findings suggested that the RNAi approach towards MMP-2 may be a potentially effective therapeutic method for the treatment of esophageal carcinoma.