Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection.

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.

Inhibition of alloresponse by a human trophoblast non-coding RNA suppressing class II transactivator promoter III and major histocompatibility class II expression in murine B-lymphocytes.

BACKGROUND: Trophoblasts are a model of natural allograft tolerance. A unique characteristic is the complete lack of expression of all classic major histocompatibility (MHC) antigens. We cloned a human trophoblast non-coding RNA (TncRNA) that suppresses MHC class II expression through inhibition of the class II transactivator (CIITA) promoter. We assessed the functional affects of TncRNA on an alloresponse and dissected the functional domain on CIITA promoter III. METHODS: Murine B-cell line A20 was transfected with TncRNA. Class II suppressed clones were selected and characterized by flow cytometry and Northern analysis. The clones were then subjected to lymphocyte proliferation assay to assess the stimulation of T-lymphocytes. CIITA promoter III-luciferase reporter plasmids were used with TncRNA plasmids in co-transfection assays; 5'-end deletion plasmids were used to dissect the promoter. RESULTS: Significant suppression of I-Ad expression was seen. Northern blot scans demonstrated 84% to 93% suppression of class II transcripts. Lymphocyte proliferation assay demonstrated a 50% and 64% inhibition of lymphocyte stimulation in the 2 clones, compared to A20 wild type. Dissection of promoter III indicated that an area between bp -152 to -107 contains the functional site of TncRNA. CONCLUSION: Human TncRNA is active across species lines and significantly inhibits allogenic response to B-cells. There is concurrent suppression of constitutive class II expression in TncRNA clones mediated through a defined region of CIITA promoter III.

Current assessment and management of spontaneous pneumomediastinum: experience in 24 adult patients.

OBJECTIVES: Spontaneous pneumomediastinum (SPM) is an uncommon, benign, self-limited disorder that usually occurs in young adults without any apparent precipitating factor or disease. The purpose of this study was to review our experience in dealing with this entity and detail a reasonable course of assessment and management. METHODS: A retrospective case series was conducted to identify adult patients with SPM who were diagnosed and treated in a single institution between 1993 and 2000. RESULTS: Twenty-four patients were identified who included 18 men and 6 women with a mean age of 17.5 years. Acute onset chest pain was the predominant symptom at presentation. Only half of the patients developed clinically evident subcutaneous emphysema. The most frequent precipitating factor was a history of illegal drug abuse seen in 25% of patients. Other factors included asthmatic bronchospasm, physical activity and violent coughing or vomiting. Chest radiography and computerized tomography (CT) were diagnostic in all cases with CT scan revealing six cases with associated pulmonary abnormalities. Esophagogram and flexible bronchoscopy were selectively used. Twelve patients (50%) were admitted to the hospital. Their mean hospital stay was 2 days. All patients were conservatively treated. In a follow-up of 3-10 years no complications or recurrences were observed. CONCLUSIONS: SPM follows alveolar rupture in the pulmonary interstitium. It shows a rising incidence in young drug users. It has a wide range of clinical features necessitating a high index of suspicion. Chest X-ray and CT scan should be always performed. Hospitalization and aggressive approach should be limited. SPM responds well to conservative treatment and follows a benign natural course.

Class II transactivator promoter activity is suppressed through regulation by a trophoblast noncoding RNA.

BACKGROUND: Trophoblasts lack expression of all classic major histocompatibility complex (MHC) antigens. Determination of the mechanism involved could provide insight into selective gene suppression and allograft tolerance. Suppression of class II expression in trophoblasts is secondary to dominant negative trans-acting factors that suppress class II transactivator (CIITA) transcription. We recently described a trophoblast-derived noncoding RNA (TncRNA) that suppresses class II expression. We examined the effects of TncRNA on the CIITA promoter, CIITA, and MHC class II expression. METHODS: HeLa clones stably transfected with TncRNA were analyzed for MHC class II and CIITA expression by fluorescence-activated cell sorting, Northern blots, and quantitative polymerase chain reaction. Activity and functional dissection of CIITA promoter IV (pIV) was assessed by transient co-transfection of promoter-reporter constructs. Methylation of pIV was assessed by Southern blots, fluorescence-activated cell sorting, and quantitative polymerase chain reaction. RESULTS: TncRNA suppressed interferon-gamma-induced human leukocyte antigen-DR and CIITA expression in HeLa cells. The mechanism involves inhibition of CIITA pIV through a defined inhibitory domain on the promoter. The mechanism does not involve methylation of the promoter. CONCLUSIONS: A novel method of CIITA suppression is described where a noncoding RNA selectively mediates the suppression of CIITA pIV possibly by complementary RNA-DNA binding to an inhibitory domain on the promoter. Selective suppression of MHC class II could have important implications in allograft tolerance and in developing class II-deficient cells or tissues for the purpose of transplantation or drug delivery systems.

Human trophoblast noncoding RNA suppresses CIITA promoter III activity in murine B-lymphocytes.

Trophoblasts lack expression of all classical MHC antigens. Determination of the mechanism involved could provide insight into selective gene suppression and allograft tolerance. Suppression of class II expression in trophoblasts is secondary to dominant negative trans-acting factors that suppress class II transactivator (CIITA) transcription. We recently described a trophoblast-derived noncoding RNA (TncRNA) that suppresses class II expression. Murine B-cells CH27 were stably transfected with TncRNA and analyzed for MHC class II and CIITA expression by FACS and Northern blots. Functional assessment of CIITA promoter III (pIII) was performed by transient transfection of promoter-reporter constructs. Methylation of pIII was assessed by Southern blots and FACS. TncRNA suppressed constitutive I-Ak and CIITA expression in murine B-cells CH27. The mechanism involves inhibition of CIITA pIII activity. The mechanism does not involve methylation of the promoter.