Novel proteomics platforms, such as the aptamer‐based SOMAscan platform, can quantify large numbers of proteins efficiently and cost‐effectively and are rapidly growing in popularity. However, comparisons to conventional immunoassays remain underexplored, leaving investigators unsure when cross‐assay comparisons are appropriate. The correlation of results from immunoassays with relative protein quantification is explored by SOMAscan. For 63 proteins assessed in two chronic obstructive pulmonary disease (COPD) cohorts, subpopulations and intermediate outcome measures in COPD Study (SPIROMICS), and COPDGene, using myriad rules based medicine multiplex immunoassays and SOMAscan, Spearman correlation coefficients range from −0.13 to 0.97, with a median correlation coefficient of ≈0.5 and consistent results across cohorts. A similar range is observed for immunoassays in the population‐based Multi‐Ethnic Study of Atherosclerosis and for other assays in COPDGene and SPIROMICS. Comparisons of relative quantification from the antibody‐based Olink platform and SOMAscan in a small cohort of myocardial infarction patients also show a wide correlation range. Finally, cis pQTL data, mass spectrometry aptamer confirmation, and other publicly available data are integrated to assess relationships with observed correlations. Correlation between proteomics assays shows a wide range and should be carefully considered when comparing and meta‐analyzing proteomics data across assays and studies.

Implementing precision medicine for complex diseases such as chronic obstructive lung disease (COPD) will require extensive use of biomarkers and an in-depth understanding of how genetic, epigenetic, and environmental variations contribute to phenotypic diversity and disease progression. A meta-analysis from two large cohorts of current and former smokers with and without COPD [SPIROMICS (N = 750); COPDGene (N = 590)] was used to identify single nucleotide polymorphisms (SNPs) associated with measurement of 88 blood proteins (protein quantitative trait loci; pQTLs). PQTLs consistently replicated between the two cohorts. Features of pQTLs were compared to previously reported expression QTLs (eQTLs). Inference of causal relations of pQTL genotypes, biomarker measurements, and four clinical COPD phenotypes (airflow obstruction, emphysema, exacerbation history, and chronic bronchitis) were explored using conditional independence tests. We identified 527 highly significant (p < 8 X 10−10) pQTLs in 38 (43%) of blood proteins tested. Most pQTL SNPs were novel with low overlap to eQTL SNPs. The pQTL SNPs explained >10% of measured variation in 13 protein biomarkers, with a single SNP (rs7041; p = 10−392) explaining 71%-75% of the measured variation in vitamin D binding protein (gene = GC). Some of these pQTLs [e.g., pQTLs for VDBP, sRAGE (gene = AGER), surfactant protein D (gene = SFTPD), and TNFRSF10C] have been previously associated with COPD phenotypes. Most pQTLs were local (cis), but distant (trans) pQTL SNPs in the ABO blood group locus were the top pQTL SNPs for five proteins. The inclusion of pQTL SNPs improved the clinical predictive value for the established association of sRAGE and emphysema, and the explanation of variance (R2) for emphysema improved from 0.3 to 0.4 when the pQTL SNP was included in the model along with clinical covariates. Causal modeling provided insight into specific pQTL-disease relationships for airflow obstruction and emphysema. In conclusion, given the frequency of highly significant local pQTLs, the large amount of variance potentially explained by pQTL, and the differences observed between pQTLs and eQTLs SNPs, we recommend that protein biomarker-disease association studies take into account the potential effect of common local SNPs and that pQTLs be integrated along with eQTLs to uncover disease mechanisms. Large-scale blood biomarker studies would also benefit from close attention to the ABO blood group.

In this report, we determined that induction of the DR a-chain gene by recombinant human interferon-y (IFN-y) in a human glioblastoma multiform cell line is transcriptionally regulated and showed that protein synthesis is not necessary for this to occur. The regions of the DR a-chain gene that are responsible for basal and recombinant IFN-y-induced gene transcription have been determined by gene transfer of a series of 5' deletion mutants in which the upstream region of the DR a chain was linked to a reporter gene, chloramphenicol acetyltransferase. Chloramphenicol acetyltransferase transcript and protein levels were determined by S1 nuclease protection and chloramphenicol acetyltransferase enzyme assays, respectively. By using these deletion mutants, we were able to draw the following conclusions. Class II major histocompatibility complex (MHC) antigens are crucial to the overall function of the immune system by virtue of their role in antigen presentation and lymphocyte interaction (1, 2). The quality of an immune response is determined in part by the timing and extent of class II antigen expression; therefore, the molecular regulation of these genes is of primary importance.The class II MHC genes are regulated in a complex fashion and provide one of the most interesting systems to study gene regulation. Expression patterns of class II antigens are extremely diverse and tightly regulated (3-8). It is assumed, therefore, that complex mechanisms must be in place that control the tissue-specific, differentiation-dependent, and inducible expression of class II genes.Several laboratories including our own have begun to examine these controlling mechanisms and to identify cisacting DNA sequences responsible for class II MHC gene regulation (9)(10)(11)(12)(13)(14)(15)(16). The present report centers on the detailed delineation of interferon-y (IFN-y)-responsive regions in the DR a-chain (DRa) gene by using a human glioblastoma multiform line as a model system. We have been particularly interested in the basal and inducible expression of these antigens on glioblastomas because class II antigen expression in the brain has been linked to the initiation and propagation of autoimmune-like diseases of the central nervous system.

We have used in vitro deletion mutagenesis in combination with DNA transfection to search for cis-acting regulatory elements involved in the tissue-specific expression of a human class H major histocompatibility complex gene. A 140-base-pair 5' flanking fragment that contains the class II box consensus sequences and an octamer sequence (AT-TTGCAT) confers tissue specificity on the promoter of the HLA-DRx gene. Recombinant DNA plasmids containing this DRa gene segment fused to the coding sequence of the bacterial chloramphenicol acetyltransferase gene are expressed at higher levels in human B-cell lines than in human T-cell lines. We have demonstrated that the most 5' of the class II boxes is essential for tissue-specific DRa promoter function. In addition, using an electrophoretic mobility shift assay to identify DNA binding proteins, we have detected binding of nuclear proteins to DNA probes containing the class II boxes and the octamer sequence. A protein that binds to the octamer is present at higher levels in nuclear extracts of B-cell lines than in other cell lines examined. This protein may be important for the tissue-specific expression of the HLA-DRx gene.Class II major histocompatibility complex (MHC) antigens are cell-surface glycoproteins that function in immune cellcell recognition and interaction. These molecules control the level of immune response to certain antigens through their role in the presentation of antigen to class II-restricted, antigen-specific T cells (1). There are at least three human class II antigens, designated DR, DP, and DQ. All class II molecules are composed of two noncovalently associated subunits. The heavy (a) chain is relatively nonpolymorphic, and the light (,B) chain is highly polymorphic.The genes coding for the class II MHC antigens are transcribed in a tissue-specific manner (2). Class II antigens are found primarily on certain cells of the immune system, including B lymphocytes, macrophages, and some activated T cells (3). Class II gene expression is differentially regulated in different cell types and is responsive to multiple regulators (4). For example, class II antigen expression on B cells is modulated by B-cell growth factor (5), whereas that on macrophages is modulated by y-interferon, a-fetoprotein, and prostaglandin E (4). y-Interferon can enhance or induce the expression of class II genes in many different cell types, including some nonlymphoid cells (6).A few studies have employed gene transfer experiments and sequence homology searches to identify cis-acting tran- In this report, we investigate the cis-acting DNA sequences that control tissue-specific expression of class II genes. To do this, we have transfected a set of recombinant plasmids containing 5' promoter deletions of the HLA-DRa gene fused to a bacterial indicator gene into human B-and T-cell lines. We then assayed extracts of transfected cells for DRacontrolled transient expression of the indicator gene. We have also used an electrophoretic mobility shift assay to detect specific bindi...

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DRa gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DRa promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. DNA sequence comparisons have revealed that all human and murine class II genes possess two short, highly conserved sequences located in the region 5' of the transcription start site (15). The length of the spacer region between the two consensus sequences is also highly conserved. These consensus sequences, referred to as the X and Y (or A and B) boxes, are putative cis-acting regulatory elements involved in the control of class II gene expression.Our laboratory was the first to report that the class II boxes are required for promoter function in a human class II gene, HLA-DRcx, and that a DNA probe containing these consensus sequences binds to a nuclear protein that exhibits wide tissue distribution (16). We have now extended this investigation by determining that the specific sites of protein-DNA contact are contained in the Y box. Mutation of these contact sites reduces promoter activity, providing evidence that protein-Y box interactions are critical for DRot promoter function. Such a correlation between protein-DNA interaction sites and promoter activity has not been previously demonstrated for human class II MHC genes.

No abstract

BackgroundAs a part of the longitudinal Chronic Obstructive Pulmonary Disease (COPD) study, Subpopulations and Intermediate Outcome Measures in COPD study (SPIROMICS), blood samples are being collected from 3200 subjects with the goal of identifying blood biomarkers for sub-phenotyping patients and predicting disease progression. To determine the most reliable sample type for measuring specific blood analytes in the cohort, a pilot study was performed from a subset of 24 subjects comparing serum, Ethylenediaminetetraacetic acid (EDTA) plasma, and EDTA plasma with proteinase inhibitors (P100™).Methods105 analytes, chosen for potential relevance to COPD, arranged in 12 multiplex and one simplex platform (Myriad-RBM) were evaluated in duplicate from the three sample types from 24 subjects. The reliability coefficient and the coefficient of variation (CV) were calculated. The performance of each analyte and mean analyte levels were evaluated across sample types.Results20% of analytes were not consistently detectable in any sample type. Higher reliability and/or smaller CV were determined for 12 analytes in EDTA plasma compared to serum, and for 11 analytes in serum compared to EDTA plasma. While reliability measures were similar for EDTA plasma and P100 plasma for a majority of analytes, CV was modestly increased in P100 plasma for eight analytes. Each analyte within a multiplex produced independent measurement characteristics, complicating selection of sample type for individual multiplexes.ConclusionsThere were notable detectability and measurability differences between serum and plasma. Multiplexing may not be ideal if large reliability differences exist across analytes measured within the multiplex, especially if values differ based on sample type. For some analytes, the large CV should be considered during experimental design, and the use of duplicate and/or triplicate samples may be necessary. These results should prove useful for studies evaluating selection of samples for evaluation of potential blood biomarkers.

BackgroundSubpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument.MethodsThe Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data.ResultsThus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel.ConclusionsOur study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters.Trial registrationThis study was registered with ClinicalTrials.gov as NCT01969344.