Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-ß and IFN-λ1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-λ, and the subtypes of IFN-λ are expressed hierarchically in the order IFN-λ1 followed by IFN-λ2, and then IFN-λ3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.

High-throughput quantitative real-time RT-PCR assay for determining expression profiles of types I and III interferon subtypes.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

True vertical validation in facial orthognathic surgery planning.

OBJECTIVES: To validate the effectiveness of the original standards of True Vertical (TV) Subnasal Line in orthognatic surgery planning. The present study evaluates the changes occurring in patients with skeletal Class II alterations programmed for orthognathic surgery with a view to improving their facial profile. STUDY DESIGN: [corrected] We showed a series of black profiles (composed by a first control group of subjects with normal occlusion, and another two additional groups comprised patients before -Group 2- and after orthognatic surgical correction of Class II malocclusion -Group 3-) for three groups of observers (orthodontists, surgeons and laypeople). The facial images became black silhouettes in order to determine a series of parameters (including aesthetic assessment) by means of the observers. Their observation were assessed using a 5-point Likert scale. RESULTS: The sample was composed of 52 profile's subjects who were tested for a total of 72 observers. Aesthetic assessment yielded mean scores of 2.57, 1.67 and 2.46 for groups 1, 2 and 3, respectively. There was a statistically significant difference (p<0.001) between group 1 versus group 2. There were no significant differences in terms of observer assessment of aesthetics, with the exception of a wider perception range among the orthodontists. Regarding the studied profile measures, significant differences were recorded for point B' and Pg' (p<0.02) between groups 2 and 3 (i.e., pre- versus post-surgery). CONCLUSIONS: The results of our study suggest the subnasale vertical and sagittal measures of the lower third of the face are decisive in facial aesthetics, and therefore also for the planning of orthognathic surgery. Consequently, these aesthetic parameters can be used as an objective tool for the planning of orthodontic treatment. Key words:Facial profile, Class II, orthognathic surgery, cephalometric analysis, facial soft tissue, subnasale vertical.

Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal RNA are often used for this purpose. Although unstable or inconsistent housekeeping gene expression will misrepresent experimental effects on target gene expression, housekeeping genes are often chosen arbitrarily rather than systematically. We designed a simple and systematic approach towards selection of housekeeping genes based on Ct variance (as reflected by the standard deviation) and normality of distribution. We validated this approach by comparing stability and consistency of expression of 11 housekeeping genes across different types of cells, experimental treatments, and human donors. Finally, we demonstrated the consequences of inconsistent housekeeping gene expression on the calculation of target gene expression, and conclude that validation of stability of housekeeping gene expression by considering both distribution normality and standard deviation is straightforward and critical for proper experimental design.