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1.  [No title available] 
PMCID: PMC3943659  PMID: 24291346
2.  [No title available] 
PMCID: PMC3955096  PMID: 24374374
3.  [No title available] 
PMCID: PMC4104170  PMID: 24316020
4.  [No title available] 
PMCID: PMC3944894  PMID: 24295867
5.  [No title available] 
PMCID: PMC3945064  PMID: 24291343
6.  Review: 2-mercaptoethanol alteration of in vitro immune functions of species other than murine 
Descriptions that organosulfurs could alter biologically relevant cellular functions began some 40 years ago when cell mediated and humoral murine in vitro immune responses were reported to be dramatically enhanced by any of four xenobiotic, sulfhydryl compounds—2-mercaptoethanol (2-ME), dithiothreitol, glutathione, and L-cysteine; the most effective of the four was 2-ME. These findings triggered a plethora of reports defining 2-ME benefits for a multitude of immunological processes, primarily with murine models. This led to investigations on 2-ME alterations of (a) immune functions in other species, (b) activities of other cell-types, and (c) in situ diseases. In addition, the early findings may have been instrumental in identification of the previously undefined anticarcinogenic chemicals in specific foods as organosulfurs. Outside the plant organosulfurs, there are no comprehensive reviews of these areas to help define mechanisms by which organosulfurs function as well as identify potential alternative uses. Therefore, the present review will focus on 2-ME alterations of in vitro immune functions in species other than murine; namely, fish, amphibian, reptile, avian, whales, dolphins, rat, hamster, rabbit, guinea pig, feline, canine, porcine, ovine, bovine, and human. Processes, some unique to a given species, were in general, enhanced and in some cases dependent upon the presence of 2-ME. The largest benefits occurred in media that were serum free, followed by those in autologous serum and then fetal bovine serum supplemented medium. Concentrations of 2-ME were generally in the low μM range, with exceptions of those for salamander (20 mM), turtles (70 mM) and dolphins (7 mM). The few studies designed to assess mechanisms found that changes induced by 2-ME were generally accompanied by alterations of reduced/oxidized glutathione cellular concentrations. The major benefit for most studies, however, was to increase the sensitivity of the culture environment, which permitted a specific process to be more easily dissected.
doi:10.1016/j.jim.2013.11.007
PMCID: PMC3946847  PMID: 24270017
2-mercaptoethanol; immune functions; non-murine species
7.  Discovery of Biomarkers for Systemic Lupus Erythematosus Using a Library of Synthetic Autoantigen Surrogates 
Journal of immunological methods  2013;402(0):23-34.
Antibodies to a wide range of self-antigens, including those directed against nucleic acids or nucleic acid-binding proteins are the essential biomarkers for diseases such as systemic lupus erythematosus (SLE). Highly complex libraries of nonamers consisting of N-substituted glycines (peptoids) were screened for compounds that bound IgG from patients with SLE and earlier, incomplete autoimmune syndromes. Peptoids were identified that could identify subjects with SLE and related syndromes with a high sensitivity (70%) and specificity (97.5%). Immobilized peptoids were used to isolate IgG from both healthy subjects and SLE patients that reacted with known RNA-binding proteins. In the case of SLE patients, the peptoid-purified IgG reacted with several autoantigens, suggesting that the peptoids are capable of interacting with multiple, structurally similar molecules. These results show that the measurement of IgG binding to peptoids can identify subjects with high levels of pathogenic autoantibodies.
doi:10.1016/j.jim.2013.11.004
PMCID: PMC3946867  PMID: 24269750
8.  Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes 
Journal of immunological methods  2013;402(0):35-42.
Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology, improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research.
doi:10.1016/j.jim.2013.11.005
PMCID: PMC4009380  PMID: 24269751
HCV; E1E2 glycoprotein; peptide array
9.  Phospho-Flow Cytometry Based Analysis of Differences in T Cell Receptor Signaling Between Regulatory T Cells and CD4+ T Cells 
CD4+ T regulatory cells (Tregs) are activated during auto-immune, injury, and inflammatory responses, however, the molecular events that trigger Treg activation are poorly understood. The purpose of this study was to investigate whether Tregs (FoxP3+ CD4+ T cells) and non-Treg CD4+ T cells might display differences in T cell receptor (TCR) dependent signaling responses following in vitro or in vivo stimulation. This study used phospho-flow cytometry as a tool to profile the kinetics and extent of TCR signaling (ZAP-70 and PKC-θ phosphorylation and expression) in Tregs and non-Tregs. We found that in vitro stimulation with anti-CD3ε induces early and transient activation of ZAP-70 and PKC-θ in both Tregs and non-Tregs. However, the response in Tregs was more rapid and higher in magnitude than responses seen in non-Tregs. In contrast, bacterial superantigen or antigen-specific TCR stimulation did not significantly activate these signaling pathways in Tregs or non-Tregs. Additional experiments tested the kinetics of in vivo TCR signaling in Tregs and non-Tregs in mice challenged with bacterial superantigen. The results of these experiments showed that superantigen rapidly activated ZAP-70 and PKC-θ in lymph node Tregs, but not in non-Tregs. In summary, we demonstrate the versatility of using phospho-flow cytometry to measure cell signaling in CD4+ T cells. The results of these in vitro and in vivo studies demonstrate that Tregs and non-Treg CD4+ T cells show marked differences in their reactivity to TCR-dependent stimulation and contribute new insights into basic mechanisms that lead to Treg activation.
doi:10.1016/j.jim.2011.08.023
PMCID: PMC4287240  PMID: 21945004
T cell activation; CD4+ T regulatory cells; T cell receptor; intracellular signaling; differential cell activation
10.  High Resolution MRI for Non-invasive Mouse Lymph Node Mapping 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.06.013.
Mouse models are fundamental to the study and design of new techniques for the cancer diagnosis and treatment. The lymphatic system plays an active role in oncogenesis and metastatic disease progression. However, the in vivo identification of LNs in mice is challenging with conventional imaging modalities since the LN diameter in normal mice is 1–2 mm. Standard dissection techniques are challenging and can only provide endpoint data. Here, we describe high resolution MRI (HR-MRI) approaches for the non-invasive detection of mouse LNs in vivo. We compare in vivo non-invasive HR-MRI methods (without exogenous contrast injections) to the ex vivo dye injection methods for the identification of commonly studied LNs in both normal mice and a mouse model of pancreatic ductal adenocarcinoma (PDAC). We demonstrated the potential to use HR-MRI techniques as a non-invasive imaging assay for visualizing mouse LNs in vivo.
doi:10.1016/j.jim.2013.06.013
PMCID: PMC3855564  PMID: 23850992
mouse; lymph node; lymphatic drainage; India ink; magnetic resonance imaging
11.  BlockLogo: visualization of peptide and sequence motif conservation 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.08.014.
BlockLogo is a web-server application for visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, selection of motif positions, type of sequence, and output format definition. The output has BlockLogo along with the sequence logo, and a table of motif frequencies. We deployed BlockLogo as an online application and have demonstrated its utility through examples that show visualization of T-cell epitopes and B-cell epitopes (both continuous and discontinuous). Our additional example shows a visualization and analysis of structural motifs that determine specificity of peptide binding to HLA-DR molecules. The BlockLogo server also employs selected experimentally validated prediction algorithms to enable on-the-fly prediction of MHC binding affinity to 15 common HLA class I and class II alleles as well as visual analysis of discontinuous epitopes from multiple sequence alignments. It enables the visualization and analysis of structural and functional motifs that are usually described as regular expressions. It provides a compact view of discontinuous motifs composed of distant positions within biological sequences. BlockLogo is available at: http://research4.dfci.harvard.edu/cvc/blocklogo/ and http://methilab.bu.edu/blocklogo/
doi:10.1016/j.jim.2013.08.014
PMCID: PMC3856553  PMID: 24001880
T-cell epitope; B-cell epitope; protein-protein interaction; block entropy; sequence variability and conservation
12.  Multiplexed VeraCode Bead-Based Serological Immunoassay for Colorectal Cancer 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.09.013.
Colorectal cancer (CRC) is the second leading cause of cancer deaths in the U.S and Western world. Despite increased screening and advances in treatment, the mortality rate (ca. 50,000/year) and high national health-care burden for CRC are likely to remain high unless an effective non-invasive screening test for CRC is instituted for a large segment of the population. Blood-based protein biomarkers hold great promise for early disease diagnosis and personalized medicine; yet robust and reproducible multiplexing platforms and methodologies have lagged behind their genomic counterparts.
Here, we report the development of a novel, multiplexed, hybrid immunoassay for CRC that is formatted on barcoded VeraCode™ micro-beads, which have until now only been used for genomic assays. The method combines a sandwich immunoassay format for detection of serum protein biomarkers with an antigen assay for autoantibody detection. The serum protein biomarkers CEA and GDF15 as well as autoantibodies to the p53 tumor associated antigen (TAA) were used to exemplify the method. This multiplex biomarker panel was configured to run on Illumina’s holographically barcoded VeraCode™ micro-bead platform, which is capable of measuring hundreds of analytes simultaneously in a single well from small volumes of blood (<50 μL) using a 96-well industry standard microtiter plate. This novel use of the VeraCode™ micro-bead platform translates into a potentially low volume, high throughput, multiplexed assay for CRC, for the purposes of biomarker validation, as well as patient screening, diagnostics and prognostics. In an evaluation of a 186 patient sera training set (CRC and normal), we obtained a diagnostic sensitivity of 54% and a specificity of 98%. We anticipate that by expanding and refining the biomarkers in this initial panel, and performing more extensive clinical validations, such an assay could ultimately provide a basis for CRC population screening to complement the more invasive, expensive and low throughput (but highly sensitive and specific) colonoscopy.
doi:10.1016/j.jim.2013.09.013
PMCID: PMC3867820  PMID: 24161315
biomarker; multiplex immunoassay; autoantibodies; tumor-associated antigens; colorectal cancer
13.  A method for high purity sorting of rare cell subsets applied to TDC 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.10.002.
TDC are a recently described subset of polyclonal αβ T-cells with dendritic cell properties. Because of their low number in peripheral immune compartments, isolation and characterization of TDC with existing purification methods is technically challenging. Here we describe a customized gating strategy and a flow cytometry-based cell sorting protocol for isolation of TDC. The protocol was developed because, despite very conservative gating for dead-cell and doublet exclusion, cells obtained with normal sorting procedures were enriched for TDC but not pure. Re-sorting the output of the first round of sorting results in highly pure TDC. Cells obtained with this method are viable and can be used for in vitro characterization. Moreover, this double-round sorting strategy can be universally applied to the isolation of other rare cell subsets.
doi:10.1016/j.jim.2013.10.002
PMCID: PMC3866963  PMID: 24120843
Flow cytometry; Sorting; TDC; Purification
14.  Adjuvant effect of diphtheria toxin after mucosal administration in both wild type and diphtheria toxin receptor engineered mouse strains 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.10.010.
The finding that murine and simian cells have differential susceptibility to diphtheria toxin (DTx) led to the development of genetically engineered mouse strains that express the simian or human diphtheria toxin receptor (DTR) under the control of various mouse gene promoters. Injection of DTx into DTR engineered mice allows for rapid and transient depletion of various cell populations. There are several advantages to this approach over global knockout mice, including normal mouse development and temporal control over when cell depletion occurs. As a result, many DTR engineered mouse strains have been developed, resulting in significant insights into the cell biology of various disease states. We used Foxp3DTR mice to attempt local depletion of Foxp3+ cells in the lung in a model of tolerance breakdown. Intratracheal administration of DTx resulted in robust depletion of lung Foxp3+ cells. However, DTx administration was accompanied by significant local inflammation, even in control C57Bl/6 mice. These data suggest that DTx administration to non-transgenic mice is not always an immunologically inert event, and proper controls must be used to assess various DTx-mediated depletion regimens.
doi:10.1016/j.jim.2013.10.010
PMCID: PMC3873768  PMID: 24200744
diphtheria toxin; lung; regulatory T cells; tolerance
15.  Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash 
Background
Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake.
Methods
Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit. Sample suitability was assessed and quality control analyses were conducted.
Results
Salivary SLPI was successfully recovered from oral gargles with low intra-assay and high inter-individual variability. Initial measurements showed that salivary SLPI varied considerably across individuals, and that SLPI was inversely associated with age.
Conclusions
This optimized assay can be used to examine the role of SLPI in the acquisition of oral HPV and other infections.
doi:10.1016/j.jim.2013.10.005
PMCID: PMC3990009  PMID: 24140751
Secretory leukocyte protease inhibitor; SLPI; Innate immunity; Immune system protein; Oral disease; Oral gargle
16.  Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys145 or Lys146 from fPSA without internal cleavages at Lys145 or Lys146 
Journal of immunological methods  2011;369(0):74-80.
Total levels of circulating prostate-specific antigen (tPSA) are strongly associated with prostate cancer (PCa) risk and outcome but benign prostate disease is the most frequent cause of a moderately elevated PSA level. Free PSA (fPSA) forms are independently associated with PCa risk and contribute modest diagnostic enhancements above and beyond tPSA alone. We developed an immunoassay for fPSA subfractions containing internal cleavages at Lys145 or Lys146 (fPSA-N). The assay was based on blocking intact single-chain fPSA (fPSA-I) with antibody 4D4 which does not detect PSA containing internal cleavages at Lys145 or Lys146. We also measured fPSA-N in blood from healthy volunteers and in anti-coagulated plasma from 76 men with or without evidence of PCa at biopsy. The analytical and functional detection limits of this assay were 0.016 ng/mL and 0.10 ng/mL, respectively. The median recovery of male fPSA-N from female plasma was 95.0 %. All 12 female samples (average age 28 years) had fPSA-N concentrations at or below the analytical detection limit. The median fPSA-N concentration (0.050 ng/mL) in 9 healthy male volunteers (age < 40 years) was below the functional detection limit, 0.420 ng/mL in 27 patients with benign prostate conditions and 0.239 ng/mL in 49 patients with PCa. Deming regression analysis of the patient samples showed that the measured fPSA-N concentrations were generally 23% lower than the previously calculated (fPSA minus fPSA-I) concentrations, likely due to differences in the antibody combinations used. In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform.
doi:10.1016/j.jim.2011.04.006
PMCID: PMC4273311  PMID: 21554885
prostate-specific antigen; free PSA isoform; internally cleaved PSA; nicked PSA; immunoassay; prostate cancer
17.  Granzyme B ELISPOT assay to measure influenza-specific cellular immunity 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.09.007.
The immunogenicity and efficacy of influenza vaccination is markedly lower in the elderly. Granzyme B (GrzB), quantified in fresh cell lysates, has been suggested to be a marker of cytotoxic T lymphocyte (CTL) response and a predictor of influenza illness among vaccinated older individuals. We have developed an influenza-specific GrzB ELISPOT assay using cryopreserved PBMCs. This method was tested on 106 healthy older subjects (age 50-74) at baseline (Day 0) and three additional time points post-vaccination (Day 3, Day 28, Day 75) with influenza A/H1N1-containing vaccine. No significant difference was seen in GrzB response between any of the time points, although influenza-specific GrzB response appears to be elevated at all post-vaccination time points. There was no correlation between GrzB response and hemagglutination inhibition (HAI) titers, indicating no relationship between the cytolytic activity and humoral antibody levels in this cohort. Additionally, a significant negative correlation between GrzB response and age was observed. These results reveal a reduction in influenza-specific GrzB response as one ages. In conclusion, we have developed and optimized an influenza-specific ELISPOT assay for use with frozen cells to quantify the CTL-specific serine protease GrzB, as a measure of cellular immunity after influenza vaccination.
doi:10.1016/j.jim.2013.09.007
PMCID: PMC3840047  PMID: 24055591
Granzyme B; cytotoxic T lymphocytes (CTL); ELISPOT; influenza virus; cellular immunity; Granzymes; Killer Cells; Natural; Perforin; T-Lymphocytes; Cytotoxic; Enzyme-Linked Immunospot Assay; Influenza; Human; Viruses; Immunuity; Cellular
18.  Development of a Diphtheria Toxin-Based Recombinant Porcine IL-2 Fusion Toxin for Depleting Porcine CD25+ Cells 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.09.006.
Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins’ ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation.
doi:10.1016/j.jim.2013.09.006
PMCID: PMC3840057  PMID: 24055128
Porcine IL-2; fusion toxin; diphtheria toxin; regulatory T cell; Pichia pastoris expression
19.  Statistical Approaches for Analyzing Immunologic Data of Repeated Observations: A Practical Guide 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.09.004.
Translational research not only encompasses transitioning from animal to human models but also must address the greater heterogeneity of humans when designing and analyzing experiments. Appropriate study designs can address heterogeneity through a priori data collection, and taking repeated measures can improve the power and efficiency of a study to detect clinically meaningful differences. Although common in other areas of biomedical research, modern statistical methods using repeated measurements on the same subject and accounting for their potential correlations are not widely utilized in immunologic studies. To highlight these analytic issues, we present a practical guide to understanding and applying analytic methods from commonly used T-tests without adjusting for multiple comparisons to mixed models with subject-specific adjustments for correlations using our data on Toll-like receptor-induced cytokine production in monocytes from young and older adults.
doi:10.1016/j.jim.2013.09.004
PMCID: PMC3840087  PMID: 24055129
Heterogeneity; mixed model; repeated measurement; multiple comparisons
20.  Endocan immunoreactivity in the mouse brain: method for identifying nonfunctional blood vessels 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.09.005.
Endocan is a secreted proteoglycan that has been shown to indicate angiogenic activity: remodeling in several tumor types in humans and mice. Serum endocan levels also indicate prognosis and has been proposed as a biomarker for certain cancers. Recently, monoclonal antibodies directed against mouse endocan have been developed allowing for further characterization of endocan function and potentially as a marker for angiogenesis through immunoreactivity in endothelial tip cells. The results of the current study show that endocan immunoreactivity in the mouse brain is present in blood vascular networks including but not limited to the cortex, hippocampus and paraventricular nucleus of the hypothalamus in C57BL/6J and FVB/N mice. Endocan immunoreactivity did not vary during postnatal development or by sex. Interestingly, after vascular perfusion with fluorescein isothiocyanate (FITC), endothelial cells positive for FITC were immunonegative for endocan suggesting FITC interference with the immunohistochemistry. A small number of FITC-negative blood vessels were endocan immunoreactive suggesting the identification of new blood vessels that are not yet functional. The current study shows that endocan is normally present in the mouse brain and prior vascular perfusion with FITC may provide a useful tool for identify newly forming blood vessels.
doi:10.1016/j.jim.2013.09.005
PMCID: PMC3840102  PMID: 24055127
endocan; Fluorescein isothiocyanate; brain
21.  A real-time killing assay to follow viral epitope presentation to CD8 T cells 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.09.009.
The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells requires the presentation of viral peptides intracellularly processed and displayed by major histocompatibility complex class I. Assays to measure CTL-mediated killing often use peptides exogenously added onto target cells –which does not account for epitope processing- or follow killing of infected cells at a single time point. In this study we established a real-time fluorogenic cytotoxic assay that measures the release of the Glucose-6-phosphate-dehydrogenase by dying target cells every 5 minutes after addition of CTL. It has comparable sensitivity to 51chromium-based killing assay with the additional advantage of incorporating the kinetics of epitope presentation. We showed that HIV infection of immortalized or primary CD4 T cells leads to asynchronous killing by two CTL clones specific for epitopes located in different proteins. Real-time monitoring of killing of virus-infected cells will enable identification of immune responses efficiently preventing virus dissemination.
doi:10.1016/j.jim.2013.09.009
PMCID: PMC3845008  PMID: 24060536
HIV; antigen processing; CD8 T cells; real-time killing assay; cytotoxicity; kinetics
22.  Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells 
Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy.
Highlights
•iTreg cells can be generated in an antigen-specific manner, even if specific Tconv cells are present at low frequency.•Blockade of anti-LFA-1 during iTreg cell differentiation augments Foxp3 induction.•The blockade of LFA-1 alters the iTreg cell phenotype but does not impair stability or function.
doi:10.1016/j.jim.2014.07.012
PMCID: PMC4265731  PMID: 25108241
Foxp3; LFA-1; Treg cell; Immunotherapy; Autoimmunity
23.  A Multiplexed Assay to Detect Antimicrobial Peptides in Biological Fluids and Cell Secretions 
Journal of immunological methods  2013;397(0):10.1016/j.jim.2013.09.001.
Mucosal tissues represent the front line in defense against potential pathogens, and one means by which mucosa provide protection is via the secretion of antimicrobials which can interfere with potential pathogens as well as recruit and modify the responses of immune cells. Here we describe adaptation of ELISA assays to microsphere format, facilitating simultaneous quantification of antimicrobial peptides including elafin, MIP3α, HBD2, HBD3, SLPI, RANTES, SDF1, lactoferrin, LL-37, and HNP1-3. The multiplexed assay exhibits excellent reproducibility, shows linearity over a two order of magnitude concentration range for most analytes, is compatible with biological fluids such as cervicovaginal lavage fluid, and presents significant cost and sample savings relative to traditional ELISA assays.
doi:10.1016/j.jim.2013.09.001
PMCID: PMC3819932  PMID: 24035708
Antimicrobials; female reproductive tract; mucosal secretions; chemokines; cytokines; multiplex assay; defensins
24.  Isolation of HIV-1-reactive antibodies using cell surface-expressed gp160ΔcBaL 
Journal of immunological methods  2013;397(0):10.1016/j.jim.2013.09.003.
Significant efforts have been made to identify HIV-1 neutralizing antibodies because they are considered to be critical to the design of an effective HIV-1 vaccine. Although soluble HIV-1 envelope proteins can be used for this purpose, these reagents differ from membrane-anchored HIV-1 envelope spike in a number of important ways and display only a subset of its native epitopes. Consistent with this, some broadly neutralizing antibodies preferentially bind cell surface-expressed HIV-1 envelope, but not the soluble protein. Here we report the details of a new method for isolating anti-HIV-1 specific B cells based on capturing cells that produce antibodies to cell surface-expressed gp160ΔcBaL. While this method is far less efficient than sorting with soluble envelope proteins, it isolated broadly neutralizing anti-HIV-1 antibodies that bind cell surface expressed gp160ΔcBaL but not soluble envelope proteins.
doi:10.1016/j.jim.2013.09.003
PMCID: PMC3854001  PMID: 24041474
HIV-1; Surface-expressed envelope trimer; Single B cell sort; HIV-1 neutralizing Antibodies
25.  Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts 
Journal of immunological methods  2013;397(0):66-70.
Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, <0.10, and 1.70 ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06 ng/mL, respectively. The second ELISA utilized mAbs 62G5 and 60G2 for capture and detection. This assay was capable of detecting 2,4- and 2,6-TDI-HSA adducts with LODs of <4.90 and 26.92 ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93 ng/mL, respectively. This 62G5-60G2 sandwich assay was also able to detect dNCO adducted transferrin, hemoglobin, keratin and actin, but with less sensitivity than dNCO-HSA. The results of this study demonstrate potential application of these ELISAs in the identification and characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures.
doi:10.1016/j.jim.2013.08.015
PMCID: PMC3907087  PMID: 24012971
Diisocyanate; Monoclonal antibody; Immunoassay; Occupational asthma

Results 1-25 (322)