Browsing by Subject "Leukocytes, Mononuclear"
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Item Open Access Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce beta-chemokines.(J Exp Med, 2010-04-12) Moody, MA; Liao, HX; Alam, SM; Scearce, RM; Plonk, MK; Kozink, DM; Drinker, MS; Zhang, R; Xia, SM; Sutherland, LL; Tomaras, GD; Giles, IP; Kappes, JC; Ochsenbauer Jambor, C; Edmonds, TG; Soares, M; Barbero, G; Forthal, DN; Landucci, G; Chang, C; King, SW; Kavlie, A; Denny, TN; Hwang, KK; Chen, PP; Thorpe, PE; Montefiori, DC; Haynes, BFTraditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.Item Open Access Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events.(PloS one, 2016-01) Buckner, Lyndsey R; Amedee, Angela M; Albritton, Hannah L; Kozlowski, Pamela A; Lacour, Nedra; McGowin, Chris L; Schust, Danny J; Quayle, Alison JChlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIVBaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.Item Open Access Development of a contemporary globally diverse HIV viral panel by the EQAPOL program.(J Immunol Methods, 2014-07) Sanchez, Ana M; DeMarco, C Todd; Hora, Bhavna; Keinonen, Sarah; Chen, Yue; Brinkley, Christie; Stone, Mars; Tobler, Leslie; Keating, Sheila; Schito, Marco; Busch, Michael P; Gao, Feng; Denny, Thomas NThe significant diversity among HIV-1 variants poses serious challenges for vaccine development and for developing sensitive assays for screening, surveillance, diagnosis, and clinical management. Recognizing a need to develop a panel of HIV representing the current genetic and geographic diversity NIH/NIAID contracted the External Quality Assurance Program Oversight Laboratory (EQAPOL) to isolate, characterize and establish panels of HIV-1 strains representing global diverse subtypes and circulating recombinant forms (CRFs), and to make them available to the research community. HIV-positive plasma specimens and previously established isolates were collected through a variety of collaborations with a preference for samples from acutely/recently infected persons. Source specimens were cultured to high-titer/high-volume using well-characterized cryopreserved PBMCs from National y donors. Panel samples were stored as neat culture supernatant or diluted into defibrinated plasma. Characterization for the final expanded virus stocks included viral load, p24 antigen, infectivity (TCID), sterility, coreceptor usage, and near full-length genome sequencing. Viruses are made available to approved, interested laboratories using an online ordering application. The current EQAPOL Viral Diversity panel includes 100 viral specimens representing 6 subtypes (A, B, C, D, F, and G), 2 sub-subtypes (F1 and F2), 7 CRFs (01, 02, 04, 14, 22, 24, and 47), 19 URFs and 3 group O viruses from 22 countries. The EQAPOL Viral Diversity panel is an invaluable collection of well-characterized reagents that are available to the scientific community, including researchers, epidemiologists, and commercial manufacturers of diagnostics and pharmaceuticals to support HIV research, as well as diagnostic and vaccine development.Item Restricted Diagnosis of partial body radiation exposure in mice using peripheral blood gene expression profiles.(PLoS One, 2010-07-12) Meadows, Sarah K; Dressman, Holly K; Daher, Pamela; Himburg, Heather; Russell, J Lauren; Doan, Phuong; Chao, Nelson J; Lucas, Joseph; Nevins, Joseph R; Chute, John PIn the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79-100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16-43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.Item Open Access Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.(Scientific reports, 2022-07-09) Giroux, Nicholas S; Ding, Shengli; McClain, Micah T; Burke, Thomas W; Petzold, Elizabeth; Chung, Hong A; Rivera, Grecia O; Wang, Ergang; Xi, Rui; Bose, Shree; Rotstein, Tomer; Nicholson, Bradly P; Chen, Tianyi; Henao, Ricardo; Sempowski, Gregory D; Denny, Thomas N; De Ussel, Maria Iglesias; Satterwhite, Lisa L; Ko, Emily R; Ginsburg, Geoffrey S; Kraft, Bryan D; Tsalik, Ephraim L; Shen, Xiling; Woods, Christopher WSARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associated with mild or moderate symptoms were already robust and included severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity was marked by upregulation of classical antiviral pathways, including those regulating IRF1 and IRF7, whereas in moderate disease, these classical antiviral signals diminished, suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.Item Open Access Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.(AIDS Res Hum Retroviruses, 2010-03) Geonnotti, Anthony R; Bilska, Miroslawa; Yuan, Xing; Ochsenbauer, Christina; Edmonds, Tara G; Kappes, John C; Liao, Hua-Xin; Haynes, Barton F; Montefiori, David CBacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.Item Open Access Early hematopoiesis inhibition under chronic radiation exposure in humans.(Radiat Environ Biophys, 2010-05) Akleyev, Alexander V; Akushevich, Igor V; Dimov, Georgy P; Veremeyeva, Galina A; Varfolomeyeva, Tatyana A; Ukraintseva, Svetlana V; Yashin, Anatoly IThe major goal of this study was to identify and quantitatively describe the association between the characteristics of chronic (low-dose rate) exposure to (low LET) ionizing radiation and cellularity of peripheral blood cell lines. About 3,200 hemograms (i.e., spectra of blood counts) obtained over the years of maximal exposure to ionizing radiation (1950-1956) for inhabitants of the Techa River were used in analyses. The mean cumulative red bone marrow dose (with standard errors), calculated using Techa River Dosimetry System-2000, was 333.6 +/- 4.6 mGy (SD = 259.9 mGy, max = 1151 mGy) to the year 1956. The statistical approach included both empirical methods for estimating frequencies of cytopenic states of the investigated blood cell lines (e.g. neutrophile, platelets, erythrocyte, etc.), and regression methods, including generalized linear models and logistic regressions which allowed taking into account confounding factors (e.g., attained age, age at maximal exposure, presence of concomitant diseases, and demographic characteristics). The results of the analyses demonstrated hematopoiesis inhibition manifested by a decrease in peripheral blood cellularity and an increase in the frequency of cytopenia in all blood cell lines (leukocytes, including lymphocytes, monocytes, neutrophiles, as well as platelets and erythrocytes). The intensity of hematopoiesis inhibition in the period of maximal exposures is determined by the combined influence of the dose rate and cumulative dose. The contribution of specific confounding factors was quantified and shown to be much less important than dose characteristics. The best predictor among dose characteristics was identified for each blood cell line. A 2-fold increase in dose rate is assumed to be a characteristic of radiosensitivity and a quantitative characteristic of the effect.Item Open Access Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials.(J Immunol Methods, 2014-07) Sambor, Anna; Garcia, Ambrosia; Berrong, Mark; Pickeral, Joy; Brown, Sara; Rountree, Wes; Sanchez, Ana; Pollara, Justin; Frahm, Nicole; Keinonen, Sarah; Kijak, Gustavo H; Roederer, Mario; Levine, Gail; D'Souza, M Patricia; Jaimes, Maria; Koup, Richard; Denny, Thomas; Cox, Josephine; Ferrari, GuidoA large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein-Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.Item Open Access Establishment of normative ranges of the healthy human immune system with comprehensive polychromatic flow cytometry profiling.(PloS one, 2019-01) Yi, John S; Rosa-Bray, Marilyn; Staats, Janet; Zakroysky, Pearl; Chan, Cliburn; Russo, Melissa A; Dumbauld, Chelsae; White, Scott; Gierman, Todd; Weinhold, Kent J; Guptill, Jeffrey TExisting normative flow cytometry data have several limitations including small sample sizes, incompletely described study populations, variable flow cytometry methodology, and limited depth for defining lymphocyte subpopulations. To overcome these issues, we defined high-dimensional flow cytometry reference ranges for the healthy human immune system using Human Immunology Project Consortium methodologies after carefully screening 127 subjects deemed healthy through clinical and laboratory testing. We enrolled subjects in the following age cohorts: 18-29 years, 30-39, 40-49, and 50-66 and enrolled cohorts to ensure an even gender distribution and at least 30% non-Caucasians. From peripheral blood mononuclear cells, flow cytometry reference ranges were defined for >50 immune subsets including T-cell (activation, maturation, T follicular helper and regulatory T cell), B-cell, and innate cells. We also developed a web tool for visualization of the dataset and download of raw data. This dataset provides the immunology community with a resource to compare and extract data from rigorously characterized healthy subjects across age groups, gender and race.Item Open Access Gene expression signatures that predict radiation exposure in mice and humans.(PLoS Med, 2007-04) Dressman, Holly K; Muramoto, Garrett G; Chao, Nelson J; Meadows, Sarah; Marshall, Dawn; Ginsburg, Geoffrey S; Nevins, Joseph R; Chute, John PBACKGROUND: The capacity to assess environmental inputs to biological phenotypes is limited by methods that can accurately and quantitatively measure these contributions. One such example can be seen in the context of exposure to ionizing radiation. METHODS AND FINDINGS: We have made use of gene expression analysis of peripheral blood (PB) mononuclear cells to develop expression profiles that accurately reflect prior radiation exposure. We demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, ranging from 50 cGy to 1,000 cGy. Likewise, a molecular signature of radiation response developed solely from irradiated human patient samples can predict and distinguish irradiated human PB samples from nonirradiated samples with an accuracy of 90%, sensitivity of 85%, and specificity of 94%. We further demonstrate that a radiation profile developed in the mouse can correctly distinguish PB samples from irradiated and nonirradiated human patients with an accuracy of 77%, sensitivity of 82%, and specificity of 75%. Taken together, these data demonstrate that molecular profiles can be generated that are highly predictive of different levels of radiation exposure in mice and humans. CONCLUSIONS: We suggest that this approach, with additional refinement, could provide a method to assess the effects of various environmental inputs into biological phenotypes as well as providing a more practical application of a rapid molecular screening test for the diagnosis of radiation exposure.Item Open Access Hematopoietic cell transplantation with cord blood for cure of HIV infections.(Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 2013-03) Petz, Lawrence D; Redei, Istvan; Bryson, Yvonne; Regan, Donna; Kurtzberg, Joanne; Shpall, Elizabeth; Gutman, Jonathan; Querol, Sergio; Clark, Pamela; Tonai, Richard; Santos, Sarah; Bravo, Aide; Spellman, Stephen; Gragert, Loren; Rossi, John; Li, Shirley; Li, Haitang; Senitzer, David; Zaia, John; Rosenthal, Joseph; Forman, Stephen; Chow, RobertHematopoietic cell transplantation (HCT) using CCR5-Δ32/Δ32 stem cells from an adult donor has resulted in the only known cure of human immunodeficiency virus (HIV) infection. However, it is not feasible to repeat this procedure except rarely because of the low incidence of the CCR5-Δ32 allele, the availability of only a small number of potential donors for most patients, and the need for a very close human leukocyte antigen (HLA) match between adult donors and recipients. In contrast, cord blood (CB) transplantations require significantly less stringent HLA matching. Therefore, our hypothesis is that cure of HIV infections by HCT can be accomplished much more readily using umbilical CB stem cells obtained from a modestly sized inventory of cryopreserved CCR5-Δ32/Δ32 CB units. To test this hypothesis, we developed a screening program for CB units and are developing an inventory of CCR5-Δ32/Δ32 cryopreserved units available for HCT. Three hundred such units are projected to provide for white pediatric patients a 73.6% probability of finding an adequately HLA matched unit with a cell dose of ≥2.5 × 10(7) total nucleated cells (TNCs)/kg and a 27.9% probability for white adults. With a cell dose of ≥1 × 10(7) TNCs/kg, the corresponding projected probabilities are 85.6% and 82.1%. The projected probabilities are lower for ethnic minorities. Impetus for using CB HCT was provided by a transplantation of an adult with acute myelogenous leukemia who was not HIV infected. The HCT was performed with a CCR5-Δ32/Δ32 CB unit, and posttransplantation in vitro studies indicated that the patient's peripheral blood mononuclear cells were resistant to HIV infection.Item Open Access Human umbilical cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications.(PloS one, 2019-01) Saha, Arjun; Patel, Sachit; Xu, Li; Scotland, Paula; Schwartzman, Jonathan; Filiano, Anthony J; Kurtzberg, Joanne; Balber, Andrew ECord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.Item Open Access Leukopak PBMC sample processing for preparing quality control material to support proficiency testing programs.(Journal of Immunological Methods, 2014-07) Garcia, Ambrosia; Keinonen, Sarah; Sanchez, Ana M; Ferrari, Guido; Denny, Thomas N; Moody, M AnthonyExternal proficiency testing programs designed to evaluate the performance of end-point laboratories involved in vaccine and therapeutic clinical trials form an important part of clinical trial quality assurance. Good clinical laboratory practice (GCLP) guidelines recommend both assay validation and proficiency testing for assays being used in clinical trials, and such testing is facilitated by the availability of large numbers of well-characterized test samples. These samples can be distributed to laboratories participating in these programs and allow monitoring of laboratory performance over time and among participating sites when results are obtained with samples derived from a large master set. The leukapheresis procedure provides an ideal way to collect samples from participants that can meet the required number of cells to support these activities. The collection and processing of leukapheresis samples require tight coordination between the clinical and laboratory teams to collect, process, and cryopreserve large number of samples within the established ideal time of ≤8 hours. Here, we describe our experience with a leukapheresis cryopreseration program that has been able to preserve the functionality of cellular subsets and that provides the sample numbers necessary to run an external proficiency testing program.Item Open Access Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.(Stem cell reviews and reports, 2015-08) Zhou, Hongyan; Martinez, Hector; Sun, Bruce; Li, Aiqun; Zimmer, Matthew; Katsanis, Nicholas; Davis, Erica E; Kurtzberg, Joanne; Lipnick, Scott; Noggle, Scott; Rao, Mahendra; Chang, StephenHuman peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.Item Open Access Screening the human exome: a comparison of whole genome and whole transcriptome sequencing.(Genome Biol, 2010) Cirulli, Elizabeth T; Singh, Abanish; Shianna, Kevin V; Ge, Dongliang; Smith, Jason P; Maia, Jessica M; Heinzen, Erin L; Goedert, James J; Goldstein, David B; Center for HIV/AIDS Vaccine Immunology (CHAVI)BACKGROUND: There is considerable interest in the development of methods to efficiently identify all coding variants present in large sample sets of humans. There are three approaches possible: whole-genome sequencing, whole-exome sequencing using exon capture methods, and RNA-Seq. While whole-genome sequencing is the most complete, it remains sufficiently expensive that cost effective alternatives are important. RESULTS: Here we provide a systematic exploration of how well RNA-Seq can identify human coding variants by comparing variants identified through high coverage whole-genome sequencing to those identified by high coverage RNA-Seq in the same individual. This comparison allowed us to directly evaluate the sensitivity and specificity of RNA-Seq in identifying coding variants, and to evaluate how key parameters such as the degree of coverage and the expression levels of genes interact to influence performance. We find that although only 40% of exonic variants identified by whole genome sequencing were captured using RNA-Seq; this number rose to 81% when concentrating on genes known to be well-expressed in the source tissue. We also find that a high false positive rate can be problematic when working with RNA-Seq data, especially at higher levels of coverage. CONCLUSIONS: We conclude that as long as a tissue relevant to the trait under study is available and suitable quality control screens are implemented, RNA-Seq is a fast and inexpensive alternative approach for finding coding variants in genes with sufficiently high expression levels.Item Open Access Setting objective thresholds for rare event detection in flow cytometry.(J Immunol Methods, 2014-07) Richards, Adam J; Staats, Janet; Enzor, Jennifer; McKinnon, Katherine; Frelinger, Jacob; Denny, Thomas N; Weinhold, Kent J; Chan, CliburnThe accurate identification of rare antigen-specific cytokine positive cells from peripheral blood mononuclear cells (PBMC) after antigenic stimulation in an intracellular staining (ICS) flow cytometry assay is challenging, as cytokine positive events may be fairly diffusely distributed and lack an obvious separation from the negative population. Traditionally, the approach by flow operators has been to manually set a positivity threshold to partition events into cytokine-positive and cytokine-negative. This approach suffers from subjectivity and inconsistency across different flow operators. The use of statistical clustering methods does not remove the need to find an objective threshold between between positive and negative events since consistent identification of rare event subsets is highly challenging for automated algorithms, especially when there is distributional overlap between the positive and negative events ("smear"). We present a new approach, based on the Fβ measure, that is similar to manual thresholding in providing a hard cutoff, but has the advantage of being determined objectively. The performance of this algorithm is compared with results obtained by expert visual gating. Several ICS data sets from the External Quality Assurance Program Oversight Laboratory (EQAPOL) proficiency program were used to make the comparisons. We first show that visually determined thresholds are difficult to reproduce and pose a problem when comparing results across operators or laboratories, as well as problems that occur with the use of commonly employed clustering algorithms. In contrast, a single parameterization for the Fβ method performs consistently across different centers, samples, and instruments because it optimizes the precision/recall tradeoff by using both negative and positive controls.Item Open Access The Center for HIV/AIDS Vaccine Immunology (CHAVI) multi-site quality assurance program for cryopreserved human peripheral blood mononuclear cells.(J Immunol Methods, 2014-07) Sarzotti-Kelsoe, Marcella; Needham, Leila K; Rountree, Wes; Bainbridge, John; Gray, Clive M; Fiscus, Susan A; Ferrari, Guido; Stevens, Wendy S; Stager, Susan L; Binz, Whitney; Louzao, Raul; Long, Kristy O; Mokgotho, Pauline; Moodley, Niranjini; Mackay, Melanie; Kerkau, Melissa; McMillion, Takesha; Kirchherr, Jennifer; Soderberg, Kelly A; Haynes, Barton F; Denny, Thomas NThe Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage.Item Open Access Therapeutic white blood cell and platelet depletions using the spectra OPTIA system continuous mononuclear cell protocol.(Journal of clinical apheresis, 2018-10) Cates, Nancy C; Oakley, Darlene J; Onwuemene, Oluwatoyosi AThe Spectra Optia apheresis system has only recently been approved by the Food and Drug Administration (FDA) for therapeutic white blood cell (WBC) depletions and is not yet approved for platelet depletions. Prior to FDA-approval of the WBC depletion protocol, when our available COBE Spectra apheresis systems were out of service, we successfully performed WBC depletion using a modified Spectra Optia apheresis system Continuous Mononuclear Cell (CMNC) protocol. Using this modified Spectra Optia CMNC protocol, we created institutional protocols for WBC and platelet depletions. We performed 10 WBC depletions in 9 patients and 2 platelet depletions in 2 patients. We compared pre- and post-procedure WBC, platelet count, and hemoglobin to the same data from patients previously treated on the COBE Spectra and found no difference in % WBC and platelet reduction. We also found no significant difference in post-procedural hematocrit decline. Additionally, adverse reactions were not increased. Therefore, we conclude that the Spectra Optia CMNC protocol can be successfully modified for effective WBC and platelet depletions without increase in adverse reactions.Item Open Access Tissue-specific genetic control of splicing: implications for the study of complex traits.(PLoS Biol, 2008-12-23) Heinzen, Erin L; Ge, Dongliang; Cronin, Kenneth D; Maia, Jessica M; Shianna, Kevin V; Gabriel, Willow N; Welsh-Bohmer, Kathleen A; Hulette, Christine M; Denny, Thomas N; Goldstein, David BNumerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes.