The Role of ADAR1 and ADAR2 in the Regulation of miRNA-21 in Idiopathic Pulmonary Fibrosis.

INTRODUCTION: microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. METHODS: Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFß1. Real-time PCR and Western blot were performed. RESULTS: ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. CONCLUSION: These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.

Profibrosing effect of angiotensin converting enzyme inhibitors in human lung fibroblasts.

OBJECTIVE: The objective of this study is to determine the effect of two angiotensin-converting enzyme inhibitors (ACEi) (Enalapril and Captopril), an angiotensin-II receptor inhibitor (Losartan) and a renin inhibitor (Aliskiren) on renin, TGF-ß1 and collagen expressions in human lung fibroblast cultures through real-time PCR and ELISA. MATERIALS AND METHODS: Normal commercial fibroblasts (CCD25) were exposed to 10(-6) M of enalapril, captopril, losartan, or aliskiren for 6 h. Subsequently, media were recovered and proteins were concentrated; RNA was extracted from the cells. Real time-PCR and ELISA were performed. RESULTS: ACEi and losartan-stimulated fibroblasts showed an increase in the expression of TGF-ß1, Collagen-Iα1 (Col-Iα1), and renin (except losartan) vs PolR2A (p < 0.05), and upregulation of TGF-ß1 protein (p < 0.01), except with aliskiren. CONCLUSION: Results show that ACEis and losartan could play a profibrosing role by inducing the overexpression of molecules such TGF-ß1 and Collagen.

Genetic susceptibility to multicase hypersensitivity pneumonitis is associated with the TNF-238 GG genotype of the promoter region and HLA-DRB1*04 bearing HLA haplotypes.

Hypersensitivity Pneumonitis (HP) is a lung inflammatory disorder caused by inhalation of organic particles by a susceptible host. Since only a small proportion of individuals exposed to HP-related antigens develop the disease, a genetic predisposition is largely suspected. However, studies regarding genetic susceptibility in this disease are scanty. We have previously found evidence supporting increased risk associated to the major histocompatibility complex (MHC) in sporadic HP. In the present study, we conducted a family-based research that includes nine multicase families with at least two related HP patients (RHP). We evaluated 19 RHP individuals, 25 additional healthy first-degree relatives (REA) and 246 healthy unrelated individuals (HUI). HLA class II typing (DRB1/3/4/5, DQA1, DQB1, DPA1, DPB1, DMA and DMB), and -863, -308 and -238 polymorphisms in the promoter region of TNF-α were performed by PCR based methods. We identified an increased frequency of HLA-DRB1*04:07, DRB1*04:05, DRB1*11:01 and DRB1*13:01 alleles in RHP individuals compared to healthy controls (p < 0.05). A significant higher frequency of DRB1*04:07-DQB1*03:02, DRB1*04:05-DQB1*03:02, and DRB1*04:03-DQB1*03:02 haplotypes was also detected in the group of patients. Likewise, TNF-238 GG genotype was more frequent in the RHP group as compared to REA (p = 0.01, OR = 7.2). Finally, the combination of HLA-DRB1*04 alleles and TNF-238 GG was significantly increased in the RHP group (p = 0.01, OR = 6.93). These findings indicate that genes located within the MHC region confer susceptibility to familial HP in Mexicans.

Up-regulation of L-selectin and E-selectin in hypersensitivity pneumonitis.

BACKGROUND: Selectins are adhesion molecules that contribute to leukocyte recruitment into the tissue after an injury. Hypersensitivity pneumonitis (HP) is a lymphocytic alveolitis, and we hypothesized that the overexpression of selectins could play a role in this process. PATIENTS AND MEASUREMENTS: We studied 16 patients with HP and 7 healthy control subjects (HCs). Sera and BAL selectins and tumor necrosis factor-alpha were determined by enzyme-linked immunosorbent assay, and cellular lung localization was determined by immunohistochemistry. Additionally, BAL L-selectin, and L-selectin-bearing T-lymphocytes analyzed by flow cytometry were evaluated in HP patients and in exposed but asymptomatic subjects (EAS). SETTING: Tertiary referral center and immunohistochemistry laboratory. RESULTS: Raised levels of E-selectin (mean [+/- SD], 178.9 +/- 30.5 vs 59.4 +/- 4.7 ng/mL, respectively; p < 0.001) and P-selectin (mean, 232.6 +/- 29.9 vs 67.6 +/- 14.2 ng/mL, respectively; p < 0.001) were detected in HP patient sera compared to control subjects, while L-selectin levels showed no differences between groups. Conversely, HP patients displayed a significant increase in levels of L-selectin found in BAL fluid compared with both HCs and EAS (11.0 +/- 1.7 vs 6.9 +/- 0.43 and 3.1 +/- 0.5 ng/mL, respectively; p < 0.05). The levels of E-selectin found in BAL fluid were similar in patients from both groups, and P-selectin was not detected. Percentage of CD3+CD62 L+ lymphocytes was lower in HP patients compared with EAS (2.33 +/- 0.8 vs 4.31 +/- 2.4, respectively; p = 0.05). By immunohistochemistry, L-selectin was detected in interstitial macrophages and polymorphonuclear cells, and E-selectin was detected in endothelial cells. CONCLUSION: These findings demonstrate that L-selectin and E-selectin are up-regulated during the development of HP, suggesting that they may contribute to the increased traffic of lung inflammatory cells.

Broncoconstricción alergica aguda en el cobayo: ventajas de la inmunización intratraqueal / Acute allergic bronchoconstriction in the guinea-pig: advantages of intratracheal immunization

De los modelos de broncoconstricción alérgica aguda el modelo animal en cobayos es el habitualmente utilizado. Sin embargo, tiene la desventaja de que la respuesta de broncoconstricción inducida por el antígeno se produce como parte de una reacción anafiláctica generalizada, situación que no ocurre en el asma de los humanos. En este trabajo se presenta un modelo inmunológico de hiperreactividad de las vías aéreas en cobayos inmunizados por vía intratraqueal. Los animales fueron sensibilizados por vía intraperitoneal o por instilación intratraqueal con ovoalbúmina (OA) y Bordetella pertussis (BP) o sólo con ovoalbúmina por vía intratraqueal. La administración intravenosa de OA a los cobayos sensibilizados produjo un aumento en la resistencia pulmonar a la inflación. No obstante en el modelo intraperitoneal los animales presentaron hipotensión severa e irreversible a dosis de l.0 y 3.l mg/kg de O.A. Este fenómeno también se observó en el modelo de inmunización intratraqueal con OA + BP pero sólo a la dosis más alta del antígeno. El estudio en úteros aislados demostró que el músculo liso de los cobayos sensibilizados por vía intraperitoneal fue 1000 veces más sensible a la ovoalbúmina que el de los cobayos inmunizados por vía respiratória. Estos datos sugieren que la inmunización local produce principalmente respuestas bronquiales