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In JoVE (1)

Other Publications (49)

Articles by Paul K. Wallace in JoVE

 JoVE Biology

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania, 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania


JoVE 4287

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Other articles by Paul K. Wallace on PubMed

Endotoxin Induces Rapid Metalloproteinase-mediated Shedding Followed by Up-regulation of the Monocyte Hemoglobin Scavenger Receptor CD163

CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI-0 indicate that a metalloproteinase is responsible for LPS-mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Advantages of Hydrophobic Culture Bags over Flasks for the Generation of Monocyte-derived Dendritic Cells for Clinical Applications

Dendritic cells (DC), potent antigen presenting cells capable of activating both naïve and primed T cells, are currently being pursued clinically in the development of cancer vaccines. Variations in the literature regarding DC source, culture conditions, maturation state, dose, and route of immunization make comparisons of clinical trial data difficult. In order to define and optimize the culture conditions for DC generation, we have performed a careful comparison of two culture methods, as well as different methods of DC maturation. Our studies demonstrate that high viability DC can be produced and matured in gas permeable hydrophobic culture bags. These cells express surface molecules characteristic of DC and have superior yield, viability, and function to cells cultured in plastic tissue culture flasks. These results suggest that hydrophobic culture bags are ideal for the preparation of clinical DC vaccines, as DC can be generated, antigen-loaded, and matured in a closed system, a scheme we have found to be superior to previously described flask culture methods.

The Pharmacokinetics of the Bispecific Antibody MDX-H210 when Combined with Interferon Gamma-1b in a Multiple-dose Phase I Study in Patients with Advanced Cancer

MDX-H210 is a Fab'xFab' bispecific antibody (BsAb) constructed chemically by crosslinking Fab' mAb 520C9 (anti-HER-2/neu) and humanized Fab' mAbH22 (anti-CD64).

Regulation and Localization of Endogenous Human Tristetraprolin

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.

Multiparameter Precursor Analysis of T-cell Responses to Antigen

Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.

A Novel Human Cancer Vaccine Elicits Cellular Responses to the Tumor-associated Antigen, Human Chorionic Gonadotropin Beta

The oncofetal antigen, human chorionic gonadotropin beta subunit (hCGbeta), is expressed by a number of carcinomas and is a prognostic indicator in renal, colorectal, bladder, and pancreatic cancers. We describe the development of a novel antibody-based dendritic cell (DC)-targeted cancer vaccine capable of eliciting cellular immune responses directed against hCGbeta.

Mannose Receptor Targeting of Tumor Antigen Pmel17 to Human Dendritic Cells Directs Anti-melanoma T Cell Responses Via Multiple HLA Molecules

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.

Use of Cell-tracking Dyes to Determine Proliferation Precursor Frequencies of Antigen-specific T Cells

The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation. The method described here uses tracking dyes to determine what proportion of T cells will proliferate in response to stimulation. As a flow cytometric "single-cell" method, it can be combined with tetramer and cytokine staining to determine the precursor frequencies of cells in the T-cell pool able to bind tetramer, to synthesize cytokines, and to proliferate in response to antigen.

Central Role of IL-6 Receptor Signal-transducing Chain Gp130 in Activation of L-selectin Adhesion by Fever-range Thermal Stress

The physiological benefit of the febrile response is poorly understood. Here we show that fever-range thermal stress enhances the function of the L-selectin lymphocyte homing receptor through an interleukin-6 (IL-6)-dependent signaling mechanism. Thermal stimulation of L-selectin adhesion in vitro and in vivo is mediated by engagement of the gp130 signal-transducing chain by IL-6 and a soluble form of the IL-6 receptor-alpha (sIL-6Ralpha) binding subunit. Thermal control of adhesion is maintained in IL-6-deficient mice through a gp130-dependent compensatory mechanism mediated by IL-6-related cytokines (i.e., oncostatin M [OSM], leukemia inhibitory factor [LIF], and IL-11). Combined biochemical and pharmacological inhibitor (PD98059, U0126, SB203580, SP600125) approaches positioned MEK1/ERK1-2, but not p38 MAPK or JNK, in the IL-6/sIL-6Ralpha signaling pathway upstream of activation of L-selectin/cytoskeletal interactions and L-selectin avidity/affinity. These results highlight a role for gp130-linked IL-6/sIL-6Ralpha transsignaling in amplifying lymphocyte trafficking during febrile inflammatory responses.

Fluorescent Human Lung Macrophages Analyzed by Spectral Confocal Laser Scanning Microscopy and Multispectral Cytometry

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.

Arsenic Trioxide Affects Signal Transducer and Activator of Transcription Proteins Through Alteration of Protein Tyrosine Kinase Phosphorylation

Arsenic trioxide decreases proliferation of acute myeloid leukemia (AML) cells, but its precise mechanism of action is unknown.

Impaired Alveolar Macrophage Response to Haemophilus Antigens in Chronic Obstructive Lung Disease

Interactions of nontypeable Haemophilus influenzae (NTHI) with macrophages are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-macrophage inflammation are poorly understood. Outer membrane protein (OMP) P6 and lipooligosaccharide (LOS) of NTHI are potent immunomodulators. We theorized that alveolar macrophages in COPD possess fundamental immune defects that permit NTHI to evade host responses.

Delineating the Mechanism by Which Selenium Deactivates Akt in Prostate Cancer Cells

The up-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is prevalent in many cancers. This phenomenon makes PI3K and Akt fruitful targets for cancer therapy and/or prevention because they are mediators of cell survival signaling. Although the suppression of phospho-Akt by selenium has been reported previously, little information is available on whether selenium modulates primarily the PI3K-phosphoinositide-dependent kinase 1 (PDK1) side of Akt phosphorylation or the phosphatase side of Akt dephosphorylation. The present study was aimed at addressing these questions in PC-3 prostate cancer cells which are phosphatase and tensin homologue-null. Our results showed that selenium decreased Akt phosphorylation at Thr308 (by PDK1) and Ser473 (by an unidentified kinase); the Thr308 site was more sensitive to selenium inhibition than the Ser473 site. The protein levels of PI3K and phospho-PDK1 were not affected by selenium. However, the activity of PI3K was reduced by 30% in selenium-treated cells, thus discouraging the recruitment of PDK1 and Akt to the membrane due to low phosphatidylinositol-3,4,5-trisphosphate formation by PI3K. Consistent with the above interpretation, the membrane localization of PDK1 and Akt was significantly diminished as shown by Western blotting. In the presence of a calcium chelator or a specific inhibitor of calcineurin (a calcium-dependent phosphatase), the suppressive effect of selenium on phospho-Akt(Ser473) was greatly reduced. The finding suggests that selenium-mediated dephosphorylation of Akt via calcineurin is likely to be an additional mechanism in regulating the status of phospho-Akt.

Antigen Presentation by Human Uterine Epithelial Cells to Autologous T Cells

Epithelial cells, as sentinels of immune protection in the endometrium, use innate immune mechanisms to protect against infection from pathogenic microbes. Our goal in this study was to assess the ability of human uterine epithelial cells to present antigen to cells of the adaptive immune system.

Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation

The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue Lavender, CellVue Plum and CellVue NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.

Cell Tracking 2007: a Proliferation of Probes and Applications

The articles in this thematic issue, entitled "Tracking Cell Proliferation and Function," illustrate some of the choices made by authors pushing the envelope for cell tracking applications in their areas of interest. Over the past decade there has been a proliferation in the range of commercially available probes for these studies, the capabilities of the instrumentation used to detect them, and in the biological systems being studied. This introductory to the thematic issue presents the advantages and limitations of the more commonly used probes such as CFSE and PKH26, as well as emerging probes that expand the range of fluorescence available, including quantum dots and the new CellVue dyes. Appropriate method and instrument setup controls and possible data analysis strategies are discussed with the goal of urging experienced investigators to include all critical information and controls when publishing their data and of aiding researchers new to cell tracking to make informed decisions on which cell tracking reagent(s) are best suited for their particular application. All cell tracking assays have the common goal of determining the fate of a particular cell population within a heterogeneous environment, whether in vivo or in vitro. Some of the common themes among the contributions found in this issue include how various probes are used to track (i) cell proliferation, (ii) regulatory and effector immune cell function and (iii) membrane transfer and antigen presentation. Although these represent only a small fraction of the large and growing list of applications for cell tracking, clearly illustrate the growing trend toward the use of multiple tracking reagents and multiple detection modalities to address complex biological questions.

A Method for Measuring Intracellular Free Magnesium Concentration in Platelets Using Flow Cytometry

Magnesium is the fourth most abundant cation in the body and is involved in over 302 enzymatic reactions. Basic science research has implicated magnesium deficiency as a cause of insulin resistance which is related to hypertension, diabetes, hyperlipidemia and increased cardiovascular risk. Research in magnesium deficiency states has been hindered because magnesium is an intracellular ion and difficult to measure. Our goal was to develop a reproducible assay to measure intracellular magnesium in platelets. Healthy volunteers agreed to have blood drawn for magnesium measurement. Platelet rich plasma was harvested from a venipuncture specimen and run through the flow cytometer. A standard titer curve using known increasing concentrations of magnesium chloride was created for each specimen, and then with the other half the specimen was run to measure the true intracellular free magnesium concentration. 15 adults agreed to volunteer for this experiment. All standard titer curves for all specimens had a correlation of > 0.99. The mean concentration of intracellular free magnesium was 450.05 microM with a range of 203.68 microM to 673.50 microM. Intracellular free magnesium can reliably and reproducibly be measured in platelets using Mag Green fluorescent dye and flow cytometry. This should advance our ability to study magnesium deficient states.

Ganciclovir Inhibits Lymphocyte Proliferation by Impairing DNA Synthesis

Cytomegalovirus (CMV) disease-related mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients has dramatically declined because of ganciclovir prophylaxis and preemptive therapeutic strategies. However, ganciclovir has not improved overall survival in randomized studies despite effectively preventing overt CMV disease. Moreover, recurrent posttransplant CMV antigenemia, associated with prolonged ganciclovir exposure, is a predictor of increased relapse of malignancy. We examined the hypothesis that ganciclovir itself may have a negative impact on immune reconstitution by testing the effect of ganciclovir on normal human lymphocytes in vitro. T-lymphocyte activation and proliferation, as measured by PHA-induced (3)H-thymidine uptake, was greatly reduced at therapeutic concentrations of ganciclovir (10 microg/mL) but not for foscarnet (300 microM/L). Moreover, ganciclovir impaired bromodeoxyuridine incorporation in proliferating lymphocytes, but did not impair lymphocyte survival or induce lymphocyte apoptosis. Collectively, these results show that ganciclovir suppresses T-lymphocyte proliferation in vitro by inhibiting DNA synthesis; with implications for T-lymphocyte function following allogeneic BMT.

Association Between CD4(+)CD25(high) T Cells and Atopy in Children

There is evidence that CD4(+)CD25(high) T-regulatory cells are important for establishing tolerance to allergens, but information in children is limited.

Rheumatoid Peripheral Blood Phagocytes Are Primed for Activation but Have Impaired Fc-mediated Generation of Reactive Oxygen Species

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fc gamma receptors (Fc gammaR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil Fc gammaRs were examined with a specific Fc gammaR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of Fc gammaRIIa and Fc gammaRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative Fc gammaR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.

Cervical Cytokine Responses in Women with Primary or Recurrent Chlamydial Infection

Little is known about concurrent expression of cervical cytokines and their regulation by sex hormones during primary or recurrent chlamydial infections in humans. Cytokine (interleukin-1beta [IL-1beta], IL-6, IL-10, interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) concentrations in cervical washes and serum samples, along with levels of beta-estradiol and progesterone in women with primary or recurrent chlamydial infections and healthy controls, were measured by ELISA. Women with recurrent infections had significantly higher levels of IFN-gamma in cervical washes than did women with primary infections. Significant negative correlation was found between IL-1beta and progesterone levels during recurrent infections. Beta-estradiol levels in women with primary infections showed significant negative correlations with cervical concentrations of IL-10, IL-1beta, and IL-6. Our study suggests that Chlamydia trachomatis infection in the female genital tract may be regulated by both the synergistic actions of the cytokines and the sex hormones beta-estradiol and progesterone.

Toll-like Receptor Activation Enhances Cell-mediated Immunity Induced by an Antibody Vaccine Targeting Human Dendritic Cells

Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGbeta). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGbeta-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

Role of Cervical Dendritic Cell Subsets, Co-stimulatory Molecules, Cytokine Secretion Profile and Beta-estradiol in Development of Sequalae to Chlamydia Trachomatis Infection

Chlamydia trachomatis infection of the female genital tract can lead to serious sequelae resulting in fertility related disorders. Little is known about the mechanism leading to Chlamydia induced pathology and factors responsible for it. As only some of the women develops reproductive disorders while majority of the women clears infection without any severe sequalae, mucosal immune response in women with or without fertility disorders was studied to identify factors which may lead to final clinical outcome of chlamydial infection.

Tracking Antigen-driven Responses by Flow Cytometry: Monitoring Proliferation by Dye Dilution

Cell-tracking reagents such as the green-fluorescent protein labeling dye CFSE and the red-fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back-calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen-specific tetramer binding, cytokine production and T-cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNgamma in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen-driven immunological responses.

Enhanced Red and Near Infrared Detection in Flow Cytometry Using Avalanche Photodiodes

Polychromatic flow cytometry enables detailed identification of cell phenotype using multiple fluorescent parameters. The photomultiplier tubes (PMTs) used to detect fluorescence in current instruments limit the sensitivity in the long wavelength spectral range. We demonstrate the flow cytometric applications of silicon avalanche photodiodes (APDs), which have improved red sensitivity and a working fluorescence detection range beyond 1,000 nm. A comparison of the wavelength-dependent performance of the APD and PMT was carried out using pulsed light-emitting diode sources, calibrated test beads, and biological samples. A breadboard flow cytometer test bench was constructed to compare the performance of PMTs and APD detectors. The APD used an additional amplifier stage to match the internal gain of the PMT. The resolution of the APD and PMT was compared for flow cytometry applications using a pulsed light-emitting diode source over the 500-1060 nm spectral range. These measurements showed the relative changes in the signal-to-noise performance of the APD and PMT over a broad spectral range. Both the APD and PMTs were used to measure the signal-to-noise response for a set of six peak calibration beads over the 530-800 nm wavelength range. CD4-positive cells labeled with antibody-conjugated phycoerythrin or 800 nm quantum dots were identified by simultaneous detection using the APD and the PMT. The ratios of the intensities of the CD4- and CD4+ populations were found to be similar for both detectors in the visible wavelengths, but only the APD was able to separate these populations at wavelengths above 800 nm. These measurements illustrate the differences in APD and PMT performance at different wavelengths and signal intensity levels. While the APD and PMT show similar signal-to-noise performance in the visible spectral range, the dark noise of the APD detector reduces the sensitivity at low signal levels. At wavelengths longer than 650 nm, the high quantum efficiency of the APD contributes to better signal-to-noise performance. The APD detector provides enhanced performance in the long wavelength region and may be used to extend the working range of the flow cytometer beyond 1,000 nm.

Increased Human Buccal Cell Autofluorescence is a Candidate Biomarker of Tobacco Smoking

Human buccal cells display diverse changes that are associated with smoked and smokeless tobacco, and clinicopathologic studies have correlated human buccal cell changes with oral cancer. Reported herein are the results of studies that were undertaken to identify a high-throughput technology that would advance efforts to use human buccal cells. We report that (a) a relatively large (mean +/- SD, 2.1 +/- 1.4 x 10(5) cells) population of human buccal cells can be collected in a noninvasive manner with a toothbrush and purified (>98% human buccal cells; n = 138 samples of the oral mucosa; n = 69 donors); (b) despite their large size (diameter, approximately 65 microm), the human buccal cells were analyzed successfully with a single laser cytometer (FACScan) and an advanced multispectral cytometer (FACSAria) having three lasers (excitation = 488, 633, and 407 nm wavelengths) and nine distinct emission channels; (c) cytometry revealed that the buccal cells expressed a high level of autofluorescence that was displayed over a broad spectrum (450-780 nm wavelength); (d) autofluorescence of human buccal cells collected from the left and right cheek was consistent, illustrating the reproducibility of the sample collection and assay procedure; (e) human buccal cell autofluorescence differed significantly among 69 adult subjects; and (f) a statistical difference (P = 0.018) between current, former, and never smokers. Summarily, this report is thought to be the first to show the application of flow cytometry for assaying human buccal cells and identifies buccal cell autofluorescence as a candidate biomarker of tobacco smoking.

Characterization of Regulatory T Cells in Urban Newborns

In the United States, asthma prevalence is particularly high among urban children. Although the underlying immune mechanism contributing to asthma has not been identified, having impaired T regulatory (Treg) cells at birth may be a determining factor in urban children. The objective of this study was to compare Treg phenotype and function in cord blood (CB) of newborns to those in peripheral blood (PB) of a subset of participating mothers.

Efficacy of Levo-1-methyl Tryptophan and Dextro-1-methyl Tryptophan in Reversing Indoleamine-2,3-dioxygenase-mediated Arrest of T-cell Proliferation in Human Epithelial Ovarian Cancer

It has been reported that levo-1-methyl tryptophan (L-1MT) can block indoleamine-2,3-dioxygenase (IDO) expressed by human dendritic cells (DC), whereas dextro-1-methyl tryptophan (D-1MT) is inefficient. However, whether L-1MT or D-1MT can efficiently reverse IDO-induced arrest of human T-cell proliferation has not been clarified. Here, we show a marked immunosuppressive effect of IDO derived from INDO-transfected 293 cell, IDO+ ovarian cancer cells, and monocyte-derived DCs on CD4+ Th1 cells, CD8+ T cells, and natural killer cells derived from peripheral blood, ascites, and tumors of ovarian cancer patients. We found that, whereas L-1MT and D/L-1MT can restore proliferation of tumor-derived and peripheral blood T-cell subsets, D-1MT does not effectively restore IDO-induced arrest of T-cell proliferation. Although D-1MT inhibited kynurenine production at high concentrations, L-1MT was more effective in abrogating kynurenine generation and tryptophan depletion, whereas tryptophan was completely depleted by IDO even in the presence of high amounts of D-1MT. Together, the results indicate that, whereas the generation of tryptophan metabolites (kynurenines) by IDO is important in mediating suppression of T-cell proliferation, the degree to which tryptophan depletion is restored by 1MT is also critical in overcoming IDO-induced arrest of T-cell proliferation.

Treating Octogenarian and Nonagenarian Acute Myeloid Leukemia Patients--predictive Prognostic Models

Treating the octogenarian and nonagenarian patients who have acute myeloid leukemia (AML) with intensive chemotherapy is controversial. Several models to predict outcome were proposed, including the use of a comorbidity index. However, it is unclear whether the Charlson comorbidity index (CCI) or the hematopoietic cell transplant comorbidity index (HCTCI) is more sensitive.

Metachronous and Synchronous Presentation of Acute Myeloid Leukemia and Lung Cancer

Smoking is associated with both acute myeloid leukemia (AML) and lung cancer. We therefore searched our database for concomitant presentation of AML and lung cancer. Among 775 AML cases and 5225 lung cancer cases presenting to Roswell Park Cancer Institute between the years January 1992 and May 2008 we found 12 (1.5% of AML cases; 0.23% of lung cancer cases) cases (seven metachronous and five synchronous) with AML and lung cancer. All but one patient were smokers. There were no unique characteristic of either AML or lung cancer in these patients. Nine patients succumbed to AML, one died from an unrelated cause while undergoing treatment for AML, one died of lung cancer and one patient is alive after allogeneic transplantation for AML. In summary, this study supports the need for effective smoking cessation programs.

Impact of Nasal Continuous Positive Airway Pressure Therapy on Markers of Platelet Activation in Patients with Obstructive Sleep Apnea

Considerable evidence implicates CD40 signaling in the pathogenesis of atheromas. Exposure to CD40 ligand induces platelet-leukocyte conjugation, a heightened expression of inflammatory cytokines, matrix-degrading enzymes, and procoagulant factors.

Validated Protocol for FoxP3 Reveals Increased Expression in Type 1 Diabetes Patients

FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes.

A Randomized Trial of Ex Vivo CD40L Activation of a Dendritic Cell Vaccine in Colorectal Cancer Patients: Tumor-specific Immune Responses Are Associated with Improved Survival

To determine whether an autologous dendritic cell (DC) vaccine could induce antitumor immune responses in patients after resection of colorectal cancer metastases and whether these responses could be enhanced by activating DCs with CD40L.

Multiparameter Flow Cytometry for the Diagnosis and Monitoring of Small GPI-deficient Cellular Populations

Glycosyl-phosphatidylinositol (GPI)-negative blood cells are diagnostic for Paroxysmal Nocturnal Hemoglobinuria (PNH). Marrow failure states are often associated with GPI-negative cell populations. Quantification of small clonal populations of GPI-negative cells influences clinical decisions to administer immunosuppressive therapy in marrow failure states (aplastic anemia or myelodysplastic syndrome) and to monitor minimal residual disease after allogeneic blood or marrow transplantation (BMT). We studied the reliability of high-resolution flow cytometry markers operating at the limits of detection.

Recurrent Deletion of 9q34 in Adult Normal Karyotype Precursor B-cell Acute Lymphoblastic Leukemia

The prognosis of adult normal karyotype (NK) precursor B-cell acute lymphoblastic leukemia (B-ALL) has not improved over the last decade, mainly because separation into distinct molecular subsets has been lacking and no targeted treatments are available. We screened the genome of blasts from 10 adult NK B-ALL patients for novel genomic alterations by array comparative genomic hybridization and verified our results with fluorescent in situ hybridization and gene expression profile with the same probes. The results demonstrate cryptic deletions of 9q34 involving SET, PKN3, NUP188, ABL1, and NUP214 in three of the samples. The smallest deletion resulted in the likely juxtaposition of the SET and NUP214 genes. This aberration has not been described before in adult NK B-ALL. Larger number of samples is warranted to determine the prognostic significance of this cryptic deletion.

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.

Long-term Safety and Efficacy of Cyclosporin A Therapy for T-cell Large Granular Lymphocyte Leukemia

Biological Effects and Clinical Significance of Lenalidomide-induced Tumour Flare Reaction in Patients with Chronic Lymphocytic Leukaemia: in Vivo Evidence of Immune Activation and Antitumour Response

Lenalidomide has demonstrated impressive antileukaemic effects in patients with chronic lymphocytic leukaemia (CLL). The mechanism(s) by which it mediates these effects remain unclear. Clinically, CLL patients treated with lenalidomide demonstrate an acute inflammatory reaction, the tumour flare reaction that is suggestive of an immune activation phenomenon. Samples from CLL patients treated with lenalidomide were used to evaluate its effect on the tumour cell and components of its microenvironment (immune cellular and cytokine). Lenalidomide was unable to directly induce apoptosis in CLL cells in vitro, however it modulated costimulatory (CD80, CD83, CD86) surface molecules on CLL cells in vitro and in vivo. Concurrently, we demonstrated that NK cell proliferation was induced by lenalidomide treatment in patients and correlated with clinical response. Cytokine analysis showed increase in levels of TNF-α post-lenalidomide treatment, consistent with acute inflammatory reaction. Furthermore, the basal cytokine profile (high IL-8, MIG, IP-10 and IL-4 levels and low IL-5, MIP1a, MIP1b, IL12/p70) was predictive of clinical response to lenalidomide. Collectively, our correlative studies provide further evidence that the antileukaemic effect of lenalidomide in CLL is mediated not only through modulation of the leukaemic clone but also through elements of the tumour microenvironment.

Tumor Flare Reaction Associated with Lenalidomide Treatment in Patients with Chronic Lymphocytic Leukemia Predicts Clinical Response

In patients with chronic lymphocytic leukemia (CLL), treatment with lenalidomide induces a unique, previously uncharacterized, immune response called tumor flare reaction (TFR). The clinical significance of this reaction remains unknown.

Treating Myelodysplastic Syndrome Improves an Accompanying Autoimmune Disease Along with a Reduction in Regulatory T-cells

Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry

In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8(+) T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses.Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells.

CD19 Expression in Acute Leukemia is Not Restricted to the Cytogenetically Aberrant Populations

Abstract Aberrant expression of the B lymphoid marker, CD19, in acute myeloid leukemia (AML) has frequently been associated with t(8;21)(q22;q22). However, AML cases lacking t(8;21) may occasionally express CD19. We asked if CD19 expression is restricted to the karyotypically abnormal leukemic cells in primary leukemia samples. We compared, by fluorescence in situ hybridization, CD19-positive and CD19-negative cells from nine acute leukemia patients: three non-t(8;21) AML, three t(8;21) AML, and three acute lymphoblastic leukemia cases. There were no significant differences in the karyotypic pattern between the CD19-positive and CD19-negative leukemic cells, raising the concern that therapeutically targeting CD19 for acute leukemia may not eradicate all malignant clones.

Predictors of Immunosuppressive Regulatory T Lymphocytes in Healthy Women

Immunosuppressive regulatory T (Treg) cells play an important role in antitumor immunity, self-tolerance, transplantation tolerance, and attenuation of allergic response. Higher proportion of Treg cells has been observed in peripheral blood of cancer cases compared to controls. Little is known about potential epidemiological predictors of Treg cell levels in healthy individuals. We conducted a cross-sectional study including 75 healthy women, between 20 and 80 years of age, who participated in the Data Bank and BioRepository (DBBR) program at Roswell Park Cancer Institute (RPCI), Buffalo, NY, USA. Peripheral blood levels of CD4(+)CD25(+)FOXP3(+) Treg cells were measured using flow cytometric analysis. A range of risk factors was evaluated using Wilcoxon Rank-Sum test, Kruskal-Wallis test, and linear regression. Age, smoking, medications for treatment of osteoporosis, postmenopausal status, body mass index (BMI), and hormone replacement therapy (HRT) were found to be significant positive predictors of Treg cell levels in peripheral blood (P ≤ 0.05). Higher education, exercise, age at first birth, oral contraceptives, and use of Ibuprofen were found be significant (P < 0.05) negative predictors of Treg levels. Thus, various epidemiological risk factors might explain interindividual variation in immune response to pathological conditions, including cancer.

Image Cytometry-based Detection of Aneuploidy by Fluorescence in Situ Hybridization in Suspension

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects.

T-cell Large Granular Lymphocytosis Associated with Malignant Thymoma

Monoallelic and Biallelic Deletions of 13q14.3 in Chronic Lymphocytic Leukemia: FISH Vs MiRNA RT-qPCR Detection

Deletion of 13q14.3 (del(13q)) is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL) and implies a favorable prognosis. We explored the feasibility of detecting del(13q) by real-time quantitative polymerase chain reaction (PCR) for miR-15a and miR-16-1, whose loci are located in the deleted region. We analyzed 23 cases of B-CLL with monoallelic (10 cases) or biallelic del(13q) (5 cases) and used trisomy 12-positive CLL samples (n = 8) as control samples. As expected, miR-15a was expressed at significantly lower levels in monoallelic del(13qx1) samples compared with trisomy 12 control samples (P = .001). Biallelic del(13q) (del(13qx2)) samples showed further reduction of miR-15a levels compared with monoallelic del(13q) (del(13qx1)) (P = .009). In contrast, miR-16-1 expression levels were generally much lower and variable, with the highest levels detected in del(13qx1). Analyzed retrospectively, miR-15a levels differ among the del(13q) groups. However, only del(13qx2) miR-15a levels are reduced enough to determine the allelic status of an individual sample prospectively by real-time quantitative PCR.

Case Study Interpretation--Portland: Case 4. Acute Leukemia of Ambiguous Lineage, Unclassifiable

Flow Cytometry As a Diagnostic Tool in Lymphomatous or Leukemic Meningitis: Ready for Prime Time?

In patients with neoplastic meningitis (NM), early diagnosis is highly desirable because the rapid institution of intrathecal therapy may mitigate the course of the disease. Cytology, long considered the "gold standard" for diagnosis, has low sensitivity because of both the paucity of cells in the cerebrospinal fluid (CSF) and morphological similarities between benign and malignant cells. A comprehensive review of the literature from 2005 through 2011 was performed that focused on diagnostic modalities for lymphomatous meningitis. Several studies demonstrated the sensitivity of flow cytometry to be several-fold higher than that of cytology for the detection of CSF leukemia/lymphoma. Patients with negative cytology but positive flow cytometry results are often asymptomatic and have lower CSF cell counts and neoplastic B-cell percentages compared with patients with positive cytology findings. Flow cytometry is a highly sensitive technique capable of accurately detecting malignant cells, even in samples with very low cell counts. Flow cytometry allows for the earlier detection of NM before the onset of clinical symptoms and CSF pleocytosis and therefore may enable more effective treatment. Future consensus regarding standardized flow cytometric antibody panels, with uniform definitions of positivity, is likely to further advance the early detection of NM by flow cytometry and permit broader clinical applicability.

Cytoreduction with Gemtuzumab Ozogamicin and Cytarabine Prior to Allogeneic Stem Cell Transplant for Relapsed/refractory Acute Myeloid Leukemia

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