Personalized cytomic assessment of vascular health: Evaluation of the vascular health profile in diabetes mellitus.

BACKGROUND: An inexpensive and accurate blood test does not currently exist that can evaluate the cardiovascular health of a patient. This study evaluated a novel high dimensional flow cytometry approach in combination with cytometric fingerprinting (CF), to comprehensively enumerate differentially expressed subsets of pro-angiogenic circulating progenitor cells (CPCs), involved in the repair of vasculature, and microparticles (MPs), frequently involved in inflammation and thrombosis. CF enabled discovery of a unique pattern, involving both MPs and CPCs and generated a personalized signature of vascular health, the vascular health profile (VHP). METHODS: Levels of CPCs and MPs were measured with a broad panel of cell surface markers in a population with atherosclerosis and type 2 diabetes mellitus (DM) and age-similar Healthy controls (HC) using an unbiased computational approach, termed CF. RESULTS: Circulating hematopoietic stem and progenitor cell (CHSPCAng) levels were detected at significantly lower concentrations in DM (P < 0.001), whereas levels of seven phenotypically distinct MPs were present at significantly higher concentrations in DM patients and one MP subset was present at significantly lower concentration in DM patients. Collectively, the combination of CHSPC(Ang) and MP levels was more informative than any one measure alone. CONCLUSIONS: This work provides the basis for a personalized cytomic vascular health profile that may be useful for a variety of applications including drug development, clinical risk assessment and companion diagnostics.

Potent obatoclax cytotoxicity and activation of triple death mode killing across infant acute lymphoblastic leukemia.

Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.

Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.(1-5) Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of: stem and progenitor cell quiescence, proliferation and/or differentiation(6-8) antigen-driven membrane transfer(9) and/or precursor cell proliferation(3,4,10-18) and immune regulatory and effector cell function(1,18-21). Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes(1,2,11). "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins(4,16,18). Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class(2-4,16,18,24). The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are: Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 10(2) - 10(3) times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile. Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Effect of darbepoetin alfa on endothelial progenitor cells and vascular reactivity in chronic kidney disease.

Endothelial progenitor cells (EPCs) are thought to be important for maintaining normal vascular function. We conducted a prospective study evaluating the effect of the erythropoiesis-stimulating agent darbepoetin alfa on EPCs and vascular function in patients with chronic kidney disease (CKD), with or without diabetes. Thirty subjects with CKD (20 subjects with type II diabetes mellitus and 10 without diabetes mellitus) received weekly subcutaneous administration of darbepoetin alfa for 4 weeks. EPCs were measured at baseline and 2 and 4 weeks after drug administration. Vascular function was measured with brachial ultrasound and cell activity was measured with a cell proliferation assay. Cells expressing CD133, CD34, CD146 and CD146/31 were significantly elevated (all p < 0.05), flow-mediated vasodilatation increased 2.1%, 95% CI: (0.4%, 3.8%) and colony-forming units increased twofold, 95% CI: (1.7, 2.3) after 4 weeks of treatment with darbepoetin alfa. Subjects with diabetes exhibited an increase in a subset of EPCs (CD133( +) and 34(+), p < 0.01 and p = 0.06, respectively), vasodilatation and proliferation. In conclusion, the administration of darbepoetin alfa for 4 weeks increased a subset of EPCs, improved endothelial function and increased cell proliferation, including those with diabetes, which is consistent with a favorable improvement in vascular health.

Diabetes reduces bone marrow and circulating porcine endothelial progenitor cells, an effect ameliorated by atorvastatin and independent of cholesterol.

Bone marrow derived endothelial progenitor cells (EPCs) are early precursors of mature endothelial cells which replenish aging and damaged endothelial cells. The authors studied a diabetic swine model to determine if induction of DM adversely affects either bone marrow or circulating EPCs and whether a HMG-CoA reductase inhibitor (statin) improves development and recruitment of EPCs in the absence of cholesterol lowering. Streptozotocin was administered to Yorkshire pigs to induce DM. One month after induction, diabetic pigs were treated with atorvastatin (statin, n = 10), ezetimibe (n = 10) or untreated (n = 10) and evaluated for number of bone marrow and circulating EPCs and femoral artery endothelial function. There was no effect of either medication on cholesterol level. One month after induction of DM prior to administration of drugs, the number of bone marrow and circulating EPCs significantly decreased (P < 0.0001) compared to baseline. Three months after DM induction, the mean proportion of circulating EPCs significantly increased in the atorvastatin group, but not in the control or ezetimibe groups. The control group showed progressive reduction in percentage of flow mediated vasodilatation (no dilatation at 3 months) whereas the atorvastatin group and ezetimibe exhibited vasodilatation, 6% and 4% respectively. DM results in significant impairment of bone marrow and circulating EPCs as well as endothelial function. The effect is ameliorated, in part, by atorvastatin independent of its cholesterol lowering effect. These data suggest a model wherein accelerated atherosclerosis seen with DM may, in part, result from reduction in EPCs which may be ameliorated by treatment with a statin.

Cytometric fingerprinting: quantitative characterization of multivariate distributions.

Recent technological advances in flow cytometry instrumentation provide the basis for high-dimensionality and high-throughput biological experimentation in a heterogeneous cellular context. Concomitant advances in scalable computational algorithms are necessary to better utilize the information that is contained in these high-complexity experiments. The development of such tools has the potential to expand the utility of flow cytometric analysis from a predominantly hypothesis-driven mode to one of discovery, or hypothesis-generating research. A new method of analysis of flow cytometric data called Cytometric Fingerprinting (CF) has been developed. CF captures the set of multivariate probability distribution functions corresponding to list-mode data and then "flattens" them into a computationally efficient fingerprint representation that facilitates quantitative comparisons of samples. An experimental and synthetic data were generated to act as reference sets for evaluating CF. Without the introduction of prior knowledge, CF was able to "discover" the location and concentration of spiked cells in ungated analyses over a concentration range covering four orders of magnitude, to a lower limit on the order of 10 spiked events in a background of 100,000 events. We describe a new method for quantitative analysis of list-mode cytometric data. CF includes a novel algorithm for space subdivision that improves estimation of the probability density function by dividing space into nonrectangular polytopes. Additionally it renders a multidimensional distribution in the form of a one-dimensional multiresolution hierarchical fingerprint that creates a computationally efficient representation of high dimensionality distribution functions. CF supports both the generation and testing of hypotheses, eliminates sources of operator bias, and provides an increased level of automation of data analysis.

Abundant anti-apoptotic BCL-2 is a molecular target in leukaemias with t(4;11) translocation.

Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti-apoptotic BCL-2 expression and target modulation were characterized in cell lines with t(4;11) and BCL-2 expression was examined in MLL and non-MLL infant/paediatric leukaemia cases by Western blot analysis and/or real-time polymerase chain reaction. Cytotoxicity of Genasensetrade mark (Oblimersen Sodium, G3139) alone or combined with cytotoxic drugs was assessed by MTT [(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays of the cell lines, applying pharmacostatistical response surface modelling of drug interactions. Apoptosis and cell cycle were evaluated by flow cytometry in RS4:11 cells. Primary leukaemias and cell lines with t(4;11) expressed abundant BCL2 mRNA and protein. Variable, sometimes substantial BCL2 mRNA was detected in other leukaemia subtypes. G3139 reduced BCL2 mRNA and protein in RS4:11 cells. The most sensitive cell line to single-agent G3139 was RS4:11. Low G3139 concentrations sensitized RS4:11 and MV4-11 cells to select anti-leukaemia cytotoxic drugs. In RS4:11 cells, combining G3139 with doxorubicin (ADR) increased active caspase 3 and TUNEL staining compared to ADR alone, indicating greater apoptosis, and G3139 increased S-phase progression. The abundant BCL-2 affords a molecular target in leukaemias with t(4;11). G3139 exhibits preclinical activity and synergy with select cytotoxic agents in RS4:11 and MV4-11 cells, and these effects occur through apoptosis.

CellVue Claret, a new far-red dye, facilitates polychromatic assessment of immune cell proliferation.

Flow cytometric analyses of immune cell proliferation, differentiation, and function are limited by the number of different fluorochromes that can be resolved simultaneously. Additional colors to expand functional analytic capability will facilitate higher dimensional analyses of heterogeneous cell populations by basic and clinical scientists. Our aim in these studies was to evaluate CellVue Claret, a fluorescent, far-red emitting, membrane intercalating dye (excitation maximum: 655 nm, emission maximum 677 nm), as an alternative and/or complementary probe to PKH26 and CFSE(1) for polychromatic studies of immune cell proliferation and function. Using a BD FACSCalibur and human peripheral blood mononuclear cells (PBMCs) from 8 different donors (2 donors studied twice), we compared CellVue Claret with the two most commonly used visible-emitting proliferation dyes, PKH26 and CFSE, in terms of: (1) compatibility with 7-Amino-actinomycin D (7-AAD) as a viability marker; (2) effect of dye labeling on lymphocyte viability; and (3) the proliferative response of CD3+ T lymphocytes from 0-96 hours as assessed by dilution of each of the 3 cell tracking dyes in cultures stimulated with anti-CD3 plus IL-2. Post-labeling recoveries and viabilities were similar for all 3 dyes, with modestly higher initial staining intensities and coefficients of variation for CellVue Claret than for CFSE or PKH26. Lymphocyte viabilities in stimulated or unstimulated cultures were also unaffected by choice of dye. Proliferative responses of viable CD3+ lymphocytes were comparable for all three dyes, whether results were reported as Proliferative Fraction (percent of cells that had divided one or more times) or as Precursor Frequency (percent of parent population that had gone on to proliferate in response to anti-CD3 plus IL-2). In summary, T cell proliferation analysis using CellVue Claret gives results equivalent to those obtained with PKH26 or CFSE, expanding the choice of proliferation dyes suitable for use in high dimensional polychromatic studies on flow cytometers with far red (633 nm-658 nm) excitation capabilities.

Flow cytometric measurement of circulating endothelial cells: the effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations.

BACKGROUND: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. METHODS: Blood was collected from young healthy subjects (N = 9, mean age 33 +/- 8 years), older healthy subjects (N = 13, mean age 66 +/- 8 years), and older subjects with PAD (N = 15, mean age 69 +/- 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later. RESULTS: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. CONCLUSIONS: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status.

Effect of acute exercise on endothelial progenitor cells in patients with peripheral arterial disease.

To determine whether exercise increases endothelial progenitor cells (EPCs) in patients with peripheral vascular disease, we developed a multi-parameter flow cytometry assay to rigorously assess EPCs and mature endothelial cells (ECs) in control subjects and patients with peripheral artery disease (PAD) subjected to graded exercise. Blood was collected from young healthy subjects (n = 9, mean age 33 years), older healthy subjects (n = 13, mean age 66 years), and older subjects with PAD (n = 15, mean age 69 years) before and 10 minutes after exercise. White blood cells were isolated and stained with a five-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33, PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion. Viable, low, side scatter singlets that were CD3-, 19-, and 33-negative were counted. While baseline levels of EPCs and ECs were similar among all subjects, young healthy subjects demonstrated significantly greater (p < 0.05) levels of progenitor cells (PCs) than older healthy and PAD subjects. Levels of EPCs and ECs tended to increase in all subjects after exercise; however, increases in PCs were only observed in young healthy and PAD subjects. Further, trends in the magnitude of change of subsets with exercise were most similar between young and PAD subjects. Our findings suggest that aging may reduce baseline circulating levels of PCs, but not EPCs or ECs, and that exercise-induced mobilization of subsets may differ depending on age and presence of PAD.

Flow-cytometric analysis of peripheral blood neutrophils: a simple, objective, independent and potentially clinically useful assay to facilitate the diagnosis of myelodysplastic syndromes.

The diagnosis of myelodysplastic syndromes (MDS) is based upon cytopenias, morphologic dysplasia, and cytogenetic abnormalities. Because morphologic dyspoiesis may be subtle, and many cases have normal cytogenetics, additional objective diagnostic tools are needed. We previously developed a novel peripheral blood neutrophil flow-cytometric (FCM) scoring system to identify patients with MDS. Here, in an analysis of 25 patients, we demonstrate that FCM abnormalities are independent of currently measured parameters in MDS, including cytopenias, marrow blast percent, and IPSS score. Importantly, FCM abnormalities were seen in 9/16 MDS patients with normal cytogenetics, suggesting that this simple, non-invasive assay could play a central role in the diagnosis of MDS.

Peripheral blood MDS score: a new flow cytometric tool for the diagnosis of myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders diagnosed using morphologic and clinical findings supported by cytogenetics. Because abnormalities may be subtle, diagnosis using these approaches can be challenging. Flow cytometric (FCM) approaches have been described; however the value of bone marrow immunophenotyping in MDS remains unclear due to the variability in detected abnormalities. We sought to refine the FCM approach by using peripheral blood (PB) to create a clinically useful tool for the diagnosis of MDS. METHODS: PB from 15 patients with MDS was analyzed by multiparametric flow cytometry using an extensive panel of monoclonal antibodies. Patterns of neutrophil antigen expression were compared with those of normal controls (n = 16) to establish light scatter and/or immunophenotypic abnormalities that correlated with MDS. A scoring algorithm was developed and validated prospectively on a blinded patient set. RESULTS: PB neutrophils from patients with MDS had lower side scatter and higher expression of CD66 and CD11a than did controls. Some MDS PB neutrophils demonstrated abnormal CD116 and CD10 expression. Because none of these abnormalities proved consistently diagnostic, we sought to increase the power of the assay by devising a scoring system to allow the association of multiple abnormalities and account for phenotypic variations. The PB MDS score differentiated patients with MDS from controls (P < 0.0001) in the test set. In a prospective validation, the PB MDS score successfully identified patients with MDS (sensitivity 73%, specificity 90%). CONCLUSIONS: FCM analysis of side scatter and only four additional immunophenotypic parameters of PB neutrophils using the PB MDS score proved more sensitive than standard laboratory approaches and may provide an additional, more reliable diagnostic tool in the identification of MDS.

Disruption of the structure of the Golgi apparatus and the function of the secretory pathway by mutants G93A and G85R of Cu, Zn superoxide dismutase (SOD1) of familial amyotrophic lateral sclerosis.

The Golgi apparatus of motor neurons (GA) is fragmented in sporadic amyotrophic lateral sclerosis (ALS), in familial ALS with SOD1 mutations, and in mice that express SOD1G93A of familial ALS, in which it was detected months before paralysis. In paralyzed transgenic mice expressing SOD1G93A or SOD1G85R, mutant proteins aggregated not only in the cytoplasm of motor neurons, but also in astrocytes and oligodendrocytes. Furthermore, aggregation of the G85R protein damaged astrocytes and was associated with rapidly progressing disease. In order to gain insight into the functional state of the fragmented GA, we examined the effects of S0D1 mutants G93A and G85R in Chinese Hamster Ovary Cells (CHO). In contrast to cells expressing the wt and G93A, the G85R expressers had no SOD1 activity. However, cells expressing both mutants, and to a lesser degree the wt, showed decreased survival, fragmentation of the GA, and dysfunction of the secretory pathway, which was assessed by measuring the amount of cell surface co-expressed CD4, a glycoprotein processed through the GA. The G93A and wt proteins were partially recovered in detergent insoluble fractions; while the recovery of G85R was minimal. Both mutants showed equal reductions of cell survival and function of the secretory pathway, in comparison to the wt and cells expressing mutant alsin, a protein found in rare cases of fALS. These results are consistent with the conclusion that the two SOD1 mutants, by an unknown mechanism, promote the dispersion of the GA and the dysfunction of the secretory pathway. This and other in vitro models of mutant SOD1 toxicity may prove useful in the elucidation of pathogenetic mechanisms.

Explant-induced reactivation of herpes simplex virus occurs in neurons expressing nuclear cdk2 and cdk4.

Herpes simplex virus (HSV) establishes productive (lytic) infections in nonneuronal cells and nonproductive (latent) infections in neurons. It has been proposed that HSV establishes latency because quiescent neurons lack cellular factors required for productive infection. It has been further proposed that these putative factors are induced following neuronal stress, as a requirement for HSV reactivation. To date, the identity of these putative cellular factors remains unknown. We have demonstrated that cyclin-dependent kinase (cdk) 1, 2, or 7 is required for HSV replication in nonneuronal cells. Interestingly, cdks 1 and 2 are not expressed in quiescent neurons but can be induced in stressed neurons. Thus, cdks may be among the cellular proteins required for HSV reactivation whose neuronal expression is differentially regulated during stress. Herein, we determined that neuronal expression of nuclear cdk2, cdk4, and cyclins E and D2 (which activate cdks 2 and 4, respectively) was induced following explant cultivation, a stressful stimulus that induces HSV reactivation. In contrast, neuronal expression of cdk7 and cytoplasmic cdk4 decreased during explant cultivation, whereas cdk3 was detected in the same small percentage of neurons before and after explant cultivation and cdks 1, 5, and 6 were not detected in neuronal cell bodies. HSV-1 reactivated specifically in neurons expressing nuclear cdk2 and cdk4, and an inhibitor specific for cdk2 inhibited HSV-1 reactivation. We conclude that neuronal levels of cdk2 are among the factors that determine the outcome of HSV infections of neurons.

Pharmacological cyclin-dependent kinase inhibitors inhibit replication of wild-type and drug-resistant strains of herpes simplex virus and human immunodeficiency virus type 1 by targeting cellular, not viral, proteins.

Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock- and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.