Amedeo Cappione

Functionally distinct subsets of human NK cells and monocyte/DC-like cells identified by coexpression of CD56, CD7, and CD4

The lack of natural killer (NK) cell-specific markers, as well as the overlap among several common surface antigens and functional properties, has obscured the delineation between NK cells and dendritic cells. Here, novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells, which lack CD7. In contrast to CD7+CD56+ NK cells, CD7(neg)CD56+ cells lack expression of NK cell-associated markers, but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7, we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells, indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally, only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore, using CD7 to separate CD56+ NK cells and CD56+ myeloid cells, we demonstrate that unlike resting CD7+CD56+ NK cells, the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells, thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo.

Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples

Biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures. Accurate qualification of such samples is paramount to downstream applications. We describe the development of an MIR spectroscopy-based analytical method offering simultaneous protein quantitation (0.25–5 mg/mL) and analysis of total lipid or detergent species, as well as the identification of other biomolecules present in biological samples. The method utilizes a hydrophilic PTFE membrane engineered for presentation of aqueous samples in a dried format compatible with fast infrared analysis. Unlike classical quantification techniques, the reported method is amino acid sequence independent and thus applicable to complex samples of unknown composition. By comparison to existing platforms, this MIR-based method enables direct quantification using minimal sample volume (2 µL); it is well-suited where repeat access and limited sample size are critical parameters. Further, accurate results can be derived without specialized training or knowledge of IR spectroscopy. Overall, the simplified application and analysis system provides a more cost-effective alternative to high-throughput IR systems for research laboratories with minimal throughput demands. In summary, the MIR-based system provides a viable alternative to current protein quantitation methods; it also uniquely offers simultaneous qualification of other components, notably lipids and detergents.

Stimulation of PBMC and Monocyte-Derived Macrophages via Toll-Like Receptor Activates Innate Immune Pathways in HIV-Infected Patients on Virally Suppressive Combination Antiretroviral Therapy

In HIV-infected, combination antiretroviral therapy (cART)-treated patients, immune activation and microbial translocation persist and associate with inadequate CD4 recovery and morbidity/mortality. We analyzed whether alterations in the toll-like receptor (TLR) pathway could be responsible for the immune hyperactivation seen in these patients. PBMC/monocyte-derived macrophages (MDMs) of 28 HIV+ untreated and 35 cART-treated patients with HIV-RNA < 40 cp/mL [20 Full Responders (FRs): CD4 ≥ 350; 15 Immunological Non-Responders (INRs): CD4 < 350], as well as of 16 healthy controls were stimulated with a panel of TLR agonists. We measured: CD4/CD8/CD14/CD38/HLA-DR/Ki67/AnnexinV/CD69/TLR4/8 (Flow Cytometry); PBMC expression of 84 TLR pathway genes (qPCR); PBMC/MDM cytokine release (Multiplex); and plasma lipopolysaccharide (LPS)/sCD14 (LAL/ELISA). PBMC/MDM from cART patients responded weakly to LPS stimulation but released high amounts of pro-inflammatory cytokines. MDM from these patients were characterized by a reduced expression of HLA-DR+ MDM and failed to expand activated HLA-DR+ CD38+ T-lymphocytes. PBMC/MDM from cART patients responded more robustly to ssRNA stimulation; this resulted in a significant expansion of activated CD38 + CD8 and the release of amounts of pro-inflammatory cytokines comparable to those seen in untreated viremic patients. Despite greater constitutive TLR pathway gene expression, PBMC from INRs seemed to upregulate only type I IFN genes following TLR stimulation, whereas PBMC from full responders showed a broader response. Systemic exposure to microbial antigens drives immune activation during cART by triggering TLRs. Bacterial stimulation modifies MDM function/pro-inflammatory profile in cART patients without affecting T-lymphocytes; this suggests translocating bacteria as selective stimulus to chronic innate activation during cART. High constitutive TLR activation is seen in patients lacking CD4 recovery, suggesting an exhausted immune milieu, anergic to further antigen encounters.

Distinguishing Latent from Active Mycobacterium tuberculosis Infection Using Elispot Assays: Looking Beyond Interferon-gamma

Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the Tuberculin Skin Test (TST). However, as IFN-gamma is produced by both fully rested and more recently activated populations of memory T cells, it is not surprising that the measurement of this cytokine alone cannot accurately distinguish Latent TB Infected (LTBI) subjects from those with active (infectious) disease. Accurate and rapid diagnosis of infectious individuals would allow medication to be properly allocated and other actions taken to more effectively curtail MTB spread. Analysis of multi-cytokine profiles ex vivo after stimulation of PBMCs from LTBI and active MTB subjects indicate the real possibility of successfully discerning these two disease states within 24 hours of a subjects blood draw. Due to the unparalleled sensitivity, low cost, and ease of use of Elispot assays, we propose that via a multiplex Elispot platform the accurate distinction of LTBI from active MTB-infected individuals is within reach.

Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays.

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.

Abstract 3483: A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples

Exosomes represent a subset of small particles secreted by many types of cells under both normal and pathological conditions. The release of microvesicles has demonstrated biological relevance; these particles act as mediators of intercellular communication both within the local microenvironment of the release site as well as systemically. Moreover, since exosomes contain RNA (messenger and miRNA) and protein (membrane-bound and cytosolic) from their cells of origin, and given that this content can be influenced by cell state, they also potentially represent a burgeoning target for biomarker discovery. Critical to understanding the physiological significance of these particles is the development of preparative techniques permitting reliable isolation of purified fractions. While numerous methods of exosome purification exist, including ultracentrifugation, immunoaffinity-based isolation using magnetic beads, precipitation, and ultrafiltration, most are plagued by sample limitations or require long and tedious workflows to achieve success. Here we present a rapid alternative method for the selective fractionation of exosomes from biological samples using an ultrafiltration device. Since the method is spin-based and dependent on size exclusion, the device has broad applications with regards to sample volume and/or type. Optimization of the protocol was aided through use of a mid infrared (MIR)-based spectroscopy platform that permits simultaneous monitoring of lysis conditions, protein quantitation, and analysis of total lipid content during exosome fractionation. Given the ultrafiltration device9s capacity for buffer exchange and sample concentration, purified fractions can be easily formatted to meet the requirements of any downstream analysis platform. To demonstrate this, resulting fractions were assayed by numerous techniques including flow cytometry, western blotting, ELISA-based assays, and electron microscopy. Citation Format: Amedeo Cappione, Sara Gutierrez, Masaharu Mabuchi, Janet Smith, Ivona Strug, Timothy Nadler. A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2014-3483

Abstract 4152: New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids.

Microvesicles, such as exosomes, are small particles secreted by many types of cells under both normal and pathological conditions such as cancer. These microparticles contain RNA and protein (both membrane-bound and cytosolic) from their cells of origin and thus represent a burgeoning resource for biomarker identification. While plasma and urine offer convenient, non-invasive access to sufficient sample volumes for screening purposes, major obstacles to this effort include the problematic issues of exosome isolation methods, the presence of over-abundant proteins or interfering cell debris, and the wide dynamic range of protein expression. It is clearly recognized in many fields that the quality of sample preparation ultimately impacts their performance in downstream analyses; this is particularly true where the target in question is a low abundant protein. Here we present an ultrafiltration-based method for the isolation of microvesicles from cell culture media as well as body fluids. To assist in process optimization, we used a novel infrared (IR) based biomolecule detection system to evaluate lysis and extraction conditions and analyze protein and lipid content. This IR system is less influenced by reducing agents and detergents than either BCA or Bradford assays. Following isolation, samples were analyzed using a rapid immunodetection technique, permitting detection of biomarkers more quickly and efficiently than ever before. Citation Format: Sara Gutierrez, Ivona Strug, Amedeo Cappione, Janet L. Smith, Masaharu Mabuchi, Timothy Nadler. New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2013-4152