Search

Immunomagnetic systems have been used for positive selection of a cellfraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citratemagnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of ~ 450nm and a Fe/lipid molar ratio of 1.52 ± 0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34- cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34 + Kg1a cells. For 106 positive cells and 100 μM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%. [ABSTRACT FROM AUTHOR]

Even though anti-PD-1/PD-L1 immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) have improved survival, a high percentage of patients still do not respond to ICIs. Myeloid-derived suppressor cells (MDSCs) are circulating cells that express PD-L1 and can infiltrate and proliferate in the tumor microenvironment, inducing immunosuppression. By evaluating changes in PD-L1 expression of live peripheral blood MDSCs, we are able to define a new PD-L1 index, useful in predicting ICI escape in NSCLC patients before initiating anti-PD-1/PD-L1 immunotherapy. In this study, a cohort of 37 NSCLC patients was prospectively analyzed, obtaining independent PD-L1 indexes. In patients with a PD-L1 index > 5.88, progressive disease occurred in 58.33% of patients [median progression-free survival (PFS) = 5.73 months; 95%CI = 2.67-20.53], showing significant differences when compared with patients with a PD-L1 index ≤ 5.88, in whom 7.69% progressed and median PFS was not reached (NR); p -value = 0.0042. Overall survival (OS) was significantly worse in patients with a high vs. low PD-L1 index (41.67% vs. 76.92%; median OS = 18.03 months, 95%CI = 6.77-25.23 vs. NR, 95%CI = 1.87-NR; p -value = 0.035). The PD-L1 index can be applied to stratify NSCLC patients according to their probability of response to ICIs at baseline. In addition to quantifying tumoral expression, this index could be used to compare nonresponse to treatment.

To achieve high-sensitivity cell measurements (<1 in 10 5  cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents., (© 2024 International Society for Advancement of Cytometry.)

Type 1 diabetes (T1D) results from a breakdown in immunological tolerance, with pivotal involvement of antigen-presenting cells. In this context, antigen-specific immunotherapies have been developed to arrest autoimmunity, such as phosphatidylserine (PS)-liposomes. However, the role of certain antigen-presenting cells in immunotherapy, particularly human macrophages (Mφ) in T1D remains elusive. The aim of this study was to determine the role of Mφ in antigen-specific immune tolerance and T1D. To that end, we evaluated Mφ ability to capture apoptotic-body mimicking PS-liposomes in mice and conducted a phenotypic and functional characterisation of four human monocyte-derived Mφ (MoMφ) subpopulations (M0, M1, M2a and M2c) after PS-liposomes uptake. Our findings in mice identified Mφ as the most phagocytic cell subset in the spleen and liver. In humans, while phagocytosis rates were comparable between T1D and control individuals, PS-liposome capture dynamics differed among Mφ subtypes, favouring inflammatory (M1) and deactivated (M2c) Mφ. Notably, high nanoparticle concentrations did not affect macrophage viability. PS-liposome uptake by Mφ induced alterations in membrane molecule expression related to immunoregulation, reduced secretion of IL-6 and IL-12, and diminished autologous T-cell proliferation in the context of autoantigen stimulation. These results underscore the tolerogenic effects of PS-liposomes and emphasize their potential to target human Mφ, providing valuable insights into the mechanism of action of this preclinical immunotherapy., Competing Interests: Declaration of competing interest MV-P and MD are co-founders of Ahead Therapeutics S.L., which aims at the clinical translation of PS-liposome immunotherapy for autoimmune diseases. LA-F, MV, DG, MS, MM-A, SR-V, and BB-Z are employees of this company. MWO works for Sartorius Stedim North America, Inc., which is in the business of selling live cell analysers and reagents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While the single-platform flow cytometric CD34+ cell counting method is the preferred choice to predict the yield of mobilized peripheral blood stem cells, most flow cytometers lack the ability of hematology counter analyzers to perform volumetric counting. However, one of the problems using reference microbeads is the vanishing counting bead phenomenon. This phenomenon results in a drop in microbeads concentration and reduces the total and relative number of beads in calibration procedures. In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads. In this study we have used a direct true volumetric counting of CD34+ cells under continuous flow pump to overcome potential drawbacks with impact in rare cell analysis. To confirm this hypothesis, we have compared the results of CD34+ cell enumeration using non-volumetric vs. volumetric systems with FC500 (Beckman Coulter) and Attune NxT (ThermoFisher) flow cytometers, respectively, in mobilized peripheral blood samples. No statistically significant differences were observed between measurements of CD34+ cells using beads, when the FC500 and Attune NxT absolute counting values were compared, or when CD34+ counts were compared on the Attune NxT, either using or not using beads. Linear regressions to study the relationship between volumetric and non-volumetric CD34+ counts confirmed the accuracy of each method. Bland-Altman test showed agreement between both methods. Our data showed that CD34+ cell enumeration using a volumetric system is comparable with current counting systems. This method represents an alternative with the advantage of the simplification of sample preparation and the reduction of the analysis subjectivity., Competing Interests: Declaration of competing interest M.D.W. and J.A.B. work for Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents. The rest of authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy.

The programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) axis is one of the most widely recognized targets for cancer immunotherapy. Importantly, PD-L1 conformational changes can hinder target binding when living cells are used. Antibody affinity, equilibrium binding, association and dissociation rates, and other affinity-related constants are fundamental to ensure target saturation. Here, PD-L1 changes in conformation and their potential impact on PD-L1 function and mutation are explored. Specifically, we present detailed flow cytometry procedures to analyze PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs). This approach can also be used to study the contribution of protein conformational changes in living cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for PD-L1 + myeloid-derived suppressor cells detection by flow cytometry Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric screening of PD-L1 + myeloid-derived suppressor cells in patients with lung cancer Support Protocol 1: Bioinformatic tools for the analysis of flow cytometric data., (© 2023 Wiley Periodicals LLC.)

Flow cytometry (FCM) enumeration of peripheral blood dendritic cells (PBDCs) is a minimally invasive procedure extremely useful for immunological studies. Numbers of PBDCs vary depending on age, lifestyle, or in pathologies like cancer, leukemia or immunodeficiencies. Conventional methods for PBDC identification by FCM involve red blood cell lysis using either formaldehyde or ammonium chloride-based solutions. This specific procedure has been widely reported to cause a detrimental effect as well as an artifactual detection of target populations. Alternatively, minimal sample perturbation assays that avoid the use of erythrolytic solutions with centrifugation steps and preserve the native cellular state are simpler and more robust than conventional methods. In this study, we aimed to evaluate how conventional FCM assays can alter dendritic cell (DC) counting when compared with minimal sample perturbation protocols, in terms of absolute cell counting, percentage and stain index (SI) of PBDC subsets. We evaluated the use of three different erythrolytic solutions (CyLyse, OptiLyse C, and Pharm Lyse) on a series of n = 20 peripheral blood specimens for conventional and plasmacytoid DCs detection as well as for leukocyte and basophil detection. Our results showed a significant reduction of leukocytes and specifically, of DCs and basophils in terms of absolute number when using erythrolytic solutions. In conclusion, our study shows that PBDC counting is heavily affected when lysing solutions are used, indicating that these stellate-shaped populations appear to be more labile., (© 2022 International Society for Advancement of Cytometry.)

Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate., (© 2022 International Society for Advancement of Cytometry.)

The challenges associated with analyzing rare cells are dependent on a series of factors, which usually require large numbers of cells per sample for successful resolution. Among these is determining the minimum number of total events needed to be acquired as defined by the expected frequency of the target cell population. The choice of markers that identify the target population, as well as the event rate and the number of aborted events/second, will also determine the statistically significant detection of rare cell events. Sample preparation is another important but often overlooked factor in rare cell analysis, and in this study we examine Poisson theory and methods to determine the effect of sample manipulation on rare cell detection. After verifying the applicability of this theory, we have evaluated the potential impact of red cell lysis on rare cell analysis, and how cell rarity can be underestimated or overestimated based on erythrolytic sensitivity or resistance of healthy leukocytes and pathological rare cells., (© 2022 International Society for Advancement of Cytometry.)

Plastic pollution is a global problem. Animals and humans can ingest and inhale plastic particles, with uncertain health consequences. Nanoplastics (NPs) are particles ranging from 1 nm to 1000 nm that result from the erosion or breakage of larger plastic debris, and can be highly polydisperse in physical properties and heterogeneous in composition. Potential effects of NPs exposure may be associated with alterations in the xenobiotic metabolism, nutrients absorption, energy metabolism, cytotoxicity, and behavior. In humans, no data on NPs absorptions has been reported previously. Given that their detection relies significantly on environmental exposure, we have prospectively studied the presence of NPs in human peripheral blood (PB). Specifically, we have used fluorescence techniques and nanocytometry, together with the staining of the lipophilic dye Nile Red (NR), to demonstrate that NPs can be accurately detected using flow cytometry.•Potential effects of nanoplastics exposure.•Fluorescence techniques and nanocytometry.•Accurate detection using flow cytometry., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.D.W. and J.A.B work for Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents. M.W.O. works for Sartorius Stedim North America, Inc., which is in the business of selling live cell analyzers and reagents. M.V-P is co-founder and CSO of Ahead Therapeutics SL, a start-up company dedicated to develop nanoparticle-based immunotherapies for autoimmune diseases. The remaining authors declare no potential conflicts of interest., (© 2023 The Author(s).)

This protocol provides instructions to improve flow cytometry analysis of marrow/peripheral blood cells by avoiding erythrolytic solutions, density gradients, and washing steps. We describe two basic approaches for identifying cell surface antigens with minimal sample perturbation, which have been successfully used to identify healthy and pathologically rare cells. The greatest advantage of these approaches is that they minimize the unwanted effect caused by sample preparation, allowing for improved study of live cells at the point of analysis. For complete details on the use and execution of this protocol, please refer to Petriz et al. (2018)., Competing Interests: M.D.W. and J.A.B. are employees of Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents. The rest of the authors declare no competing interests., (© 2021 The Author(s).)

Current methods for the determination of cell-mediated cytotoxic activity in blood samples usually isolate peripheral blood mononuclear cells by density gradient centrifugation or alternatively use erythrocyte lysis. Both centrifugation and red cell lysis can cause cellular depletion and cell dysfunction, resulting in erroneous measurements. To address limitations of current assays, we developed an improved strategy to determine cellular cytotoxicity using flow cytometry. Viable nucleic acid stains are used to identify live nucleated cells and discriminate them from non-nucleated erythrocytes, platelets, and debris while avoiding lysing and washing steps to maintain cell functionality. To detect target cells, we have used two different labeling approaches. In the first approach, EGFP-labeled K562 human chronic myelogenous leukemia cells provide a "ready-to-use" target without the need of additional for labeling or staining. For the second approach, we perform parallel cytotoxicity assays in the presence of wild-type K562 cells previously loaded with a fluorescent dye that has spectral properties similar to those of EGFP. Given the importance of cytotoxic assays and the deleterious effects of current sample preparation methods, the aim of this study was to adapt this "untouched cells" flow cytometry method to study cytotoxic activity using unlysed whole blood samples and fluorescent target cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for cell-mediated cytotoxic activity determination in unlysed whole blood Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of cell-mediated cytotoxic activity using unlysed whole blood samples Support Protocol 1: Optimization of the performance of target cell labeling approaches Support Protocol 2: Assessment of the linearity and reproducibility of cytotoxicity assays., (© 2021 Wiley Periodicals LLC.)

Introduction: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice., Methods: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls., Results: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good., Conclusions: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies., (© 2020 International Clinical Cytometry Society.)

For several decades, cell-mediated cytotoxicity has been measured using the 51 Cr release assay. This assay, however, has several drawbacks and flow cytometry has been used as an alternative to measure cytotoxic activity. Here, we present a quantitative method for cell-mediated cytotoxicity studies, preserving cellular function with minimal sample manipulation. Cytotoxic activity is simply and reproducibly measured as the ability of cytotoxic cells to lyse K562 target cells previously loaded with Calcein-AM vital stain. After spiking a known number of fluorescent viable K562 target cells into whole blood, cell mixtures are incubated for 2 h in a cell incubator and the remaining spiked cells are counted by flow cytometry. In order to discriminate nucleated cells, erythrocytes, and debris, unlysed whole blood is stained with a cell permeable DNA vital fluorescent dye. Cell-mediated lysis is measured by comparing target counts for different effector-to-target ratios. Since the cytotoxicity of these dyes is relatively low, this method can be broadly applied to studies of innate immune response to tumors and infections, especially where target-killing activity might be compromised by small volume samples or low frequency of cytotoxic cells. © 2020 International Society for Advancement of Cytometry., (© 2020 International Society for Advancement of Cytometry.)

Translational research has improved the diagnosis and follow-up of hematological diseases and malignancies. However, some classical diagnostics used for research and clinical practice that have remain practically unchanged for decades may be better addressed through advances in flow cytometry technology, whereby more precise measurements may be implemented in a straightforward manner. The current method for semiquantitative analysis of granulocytic alkaline phosphatase (GAP) activity is still based on observer-dependent color-intensity classification. Here, we describe a novel strategy for flow cytometric quantification of GAP activity in which staining and analytical flow cytometry facilitate the detection and quantification of subpopulations of leukocytes with different GAP activities. Our experiments demonstrate the potential of flow cytometry as a simple and highly sensitive approach for measuring GAP activity in unlysed whole blood. Notably, a comparison of flow cytometry and enzyme cytochemistry techniques showed that enzyme activity scores were not similar, indicating that results needs to be interpreted with caution, given that the enzyme-substrate binding affinities may differ, as well as the subjective evaluation of the intensity of the precipitated dye. © 2020 Wiley Periodicals LLC. Basic Protocol: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of alkaline phosphatase activity in granulocytes from whole blood samples Support Protocol 1: Sample preparation for granulocyte alkaline phosphatase determination by flow cytometry using no-lyse no-wash methods Support Protocol 2: Data analysis and formula to calculate the GAP score., (© 2020 Wiley Periodicals LLC.)

In this prospective hospital-based cohort study that included 43 newly diagnosed patients with acute myeloid leukemia, flow cytometric cellular alkaline phosphatase (ALP) activity within primitive leukemic cells allowed us to identify two groups of patients at diagnosis according to the numbers of leukemic blasts expressing ≥ 12% of ALP+ cells (27 patients, Group A) and less than 12% of ALP+ cells (16 patients, Group B). Differences in outcome for complete response, relapse or treatment resistance, and exitus were statistically analyzed and were significant, when comparing the two groups. The overall survival (OS) and event-free survival (EFS) differences between Group A and B were statistically significant. The survival of Group A patients was significantly shorter than those for Group B. No significant relationship was detected in outcome when comparing ELN prognostic-risk group based on cytogenetic and molecular profile (patients in the favorable, intermediate, and adverse risk groups). Flow cytometric cellular ALP activity at diagnosis may be used to estimate relapses and disease persistence more accurately. The limitations of our study include the small number of patients enrolled and a short follow-up, due to its prospective nature., Competing Interests: CONFLICTS OF INTEREST M.D.W. and J.A.B. work for Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

[This corrects the article DOI: 10.18632/oncotarget.12497.].

Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1-2 µL starting blood volumes.

Red blood cell lysis is an integral part of many flow cytometry protocols. It's potential to cause artifacts has been known for decades, but lysis free sample preparation has failed to replace lysis in most applications. Studies of various lysing protocols on cell losses and effects on phenotypic markers and cell function began early in the history of immunophenotyping and continue to this day. Opportunities to combine live cell response and functional assessment with phenotyping have sparked increasing interest in no lyse no wash protocols, with minimizing sample preparation effects on the cell biology as the primary goal. No lyse no wash protocols reduce sample handling and are procedurally less complex than lysis protocols, but the impact of keeping intact red blood cells that grossly outnumber the target white blood cells, must be understood to fully take advantage of this simplicity. Presented here are theories and methods for executing and interpreting no lyse no wash assays in whole blood. Methods for distinguishing white blood cells and platelets from red blood cells and improving scatter data by combining 405 nm and 488 nm side scatter are shown. Methods for assessing white blood cell light scattering profiles for individual instruments and sample treatments are discussed within the context of example profiles for no lysis and hypotonic and ammonium chloride lysis treatments. The utility of overcoming no lyse no wash scatter and fluorescence background limitations using alternate scatter and fluorescence thresholding strategies is also discussed in the context of application examples., (Copyright © 2017. Published by Elsevier Inc.)

With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALPhigh cells appear to sustain leukemogenesis over time.

Unlabelled: Adipose tissue is a major source of mesenchymal stem cells (MSCs), which possess a variety of properties that make them ideal candidates for regenerative and immunomodulatory therapies. Here, we compared the immunophenotypic profile of human adipose-derived stem cells (hASCs) from lean and obese individuals, and explored its relationship with the apparent altered plasticity of hASCs. We also hypothesized that persistent hypoxia treatment of cultured hASCs may be necessary but not sufficient to drive significant changes in mature adipocytes. hASCs were obtained from subcutaneous adipose tissue of healthy, adult, female donors undergoing abdominal plastic surgery: lean (n=8; body mass index [BMI]: 23±1 kg/m2) and obese (n=8; BMI: 35±5 kg/m2). Cell surface marker expression, proliferation and migration capacity, and adipogenic differentiation potential of cultured hASCs at two different oxygen conditions were studied. Compared with lean-derived hASCs, obese-derived hASCs demonstrated increased proliferation and migration capacity but decreased lipid droplet accumulation, correlating with a higher expression of human leukocyte antigen (HLA)-II and cluster of differentiation (CD) 106 and lower expression of CD29. Of interest, adipogenic differentiation modified CD106, CD49b, HLA-ABC surface protein expression, which was dependent on the donor's BMI. Additionally, low oxygen tension increased proliferation and migration of lean but not obese hASCs, which correlated with an altered CD36 and CD49b immunophenotypic profile. In summary, the differences observed in proliferation, migration, and differentiation capacity in obese hASCs occurred in parallel with changes in cell surface markers, both under basal conditions and during differentiation. Therefore, obesity is an important determinant of stem cell function independent of oxygen tension., Significance: The obesity-related hypoxic environment may have latent effects on human adipose tissue-derived mesenchymal stem cells (hASCs) with potential consequences in mature cells. This study explores the immunophenotypic profile of hASCs obtained from lean and obese individuals and its potential relationship with the altered plasticity of hASCs observed in obesity. In this context, an altered pattern of cell surface marker expression in obese-derived hASCs in both undifferentiated and differentiated stages is demonstrated. Differences in proliferation, migration, and differentiation capacity of hASCs from obese adipose tissue correlated with alterations in cell surface expression. Remarkably, altered plasticity observed in obese-derived hASCs was maintained in the absence of hypoxia, suggesting that these cells might be obesity conditioned., (©AlphaMed Press.)

Introduction: Studies are needed to understand the role of CD34 expressing cells with regard to efficient engraftment, especially in the adjuvant treatment of cancer., Materials and Methods: In this study we have used a modified method in our laboratory for routinely counting CD34+ cells. Unlysed whole blood samples were stained with the DNA-selective and cell membrane-permeant Vibrant DyeCycle Violet stain., Results: CD34+ cells exhibit a consistent and differential Vybrant Dye Cycle Violet staining pattern. Based on their different DCV intensity, we classified these subpopulations as CD34+/DCV(high) and CD34+/DCV(low) cells. In general, DCV(high) cells are about 12-times brighter than DCV(low) cells., Conclusion: DCV staining may be used to discriminate subsets of CD34+ cells similarly to other methods which have previously defined different functional properties that can be related to the characterization, resolution, and purification of primitive hematopoietic stem cells in combination with specific useful markers for multicolor flow cytometric measurements.

Our goal was to address if intermittent hypobaric hypoxia (IHH) exposure can help to increase the number of peripheral blood circulating progenitor cells and side population (SP) stem cells, in order to establish the usefulness of this intervention for skeletal muscle repair, because these cells play a role in tissue regeneration. Male Sprague-Dawley rats were studied in two basal states: untrained and trained and compared with 1, 3, 7 and 14 days stages of damage recovery of trained rats that had suffered skeletal muscle injury. Three experimental groups were studied: rats with passive recovery (CTRL); rats exposed to IHH after muscle damage (HYP); and, trained rats that, in addition to IHH, performed light aerobic exercise sessions (EHYP). We observed an increase in hematopoietic stem cells (HSCs) (mean = 0.153% of cells) and endothelial progenitor cells (EPCs) (mean = 0.0020% of cells) in EHYP on day 7. Also these cells showed characteristics of more primitive progenitors in comparison to the other experimental groups (mean = 0.107% of cells), as deduced by retention of the promising fluorescent probe Vybrant Dye Cycle Violet. We concluded that intermittent exposure to hypobaric hypoxia in combination with light aerobic exercise increased the number of HSCs and EPCs on the 7th day in EHYP group, although the exercise-induced stimulus showed a reverse effect on SP kinetics.

Background: In chronic obstructive pulmonary disease (COPD), decreased progenitor cells and impairment of systemic vascular function have been suggested to confer higher cardiovascular risk. The origin of these changes and their relationship with alterations in the pulmonary circulation are unknown., Objectives: To investigate whether changes in the number of circulating hematopoietic progenitor cells are associated with pulmonary hypertension or changes in endothelial function., Methods: 62 COPD patients and 35 controls (18 non-smokers and 17 smokers) without cardiovascular risk factors other than cigarette smoking were studied. The number of circulating progenitors was measured as CD45(+)CD34(+)CD133(+) labeled cells by flow cytometry. Endothelial function was assessed by flow-mediated dilation. Markers of inflammation and angiogenesis were also measured in all subjects., Results: Compared with controls, the number of circulating progenitor cells was reduced in COPD patients. Progenitor cells did not differ between control smokers and non-smokers. COPD patients with pulmonary hypertension showed greater number of progenitor cells than those without pulmonary hypertension. Systemic endothelial function was worse in both control smokers and COPD patients. Interleukin-6, fibrinogen, high sensitivity C-reactive protein, vascular endothelial growth factor and tumor necrosis factor were increased in COPD. In COPD patients, the number of circulating progenitor cells was inversely related to the flow-mediated dilation of systemic arteries., Conclusions: Pulmonary and systemic vascular impairment in COPD is associated with cigarette smoking but not with the reduced number of circulating hematopoietic progenitors. The latter appears to be a consequence of the disease itself not related to smoking habit.

Introduction: In 1996, Goodell et al. first described a rare subpopulation of bone marrow stem cells termed the Side Population (SP). SP cells are known to be CD34 negative and to have a high repopulating capability. The SP was identified by ultraviolet excitation based on the efflux of the DNA binding dye, Hoechst 33342 (Ho342). ABCG2, a halftransporter that belongs to the ATP binding cassette transporter superfamily, is the major contributor to the SP phenotype by actively pumping Ho432 selectively from stem cells. To date, very little is known about the identification of the SP in peripheral blood samples, and about its peripheral circulation, enrichment or isolation to evaluate its therapeutic potential. Due to the SP potential role in tissue regeneration, we studied the numbers of the SP in bone marrow and peripheral blood samples in regard to count accuracy and reproducibility., Materials and Methods: Bone marrow (BM) and apheresis (AP) specimens were obtained from healthy donors and patients undergoing stem cell transplantation. Bone marrow samples were obtained by aspiration. Peripheral blood cells after granulocyte colony stimulating factor (G-CSF) mobilization with or without chemotherapy, were obtained by apheresis. All samples were prepared for identification of SP cells by flow cytometry., Results: SP cells were detected in only 19 of 111 apheretic products, with relative frequency ranging from 0.01 to 4.75% of cells by the Ho342 exclusion method and flow cytometry analysis. Cell preparations used for these measurements consisted of 5 x 10(6) cells. However, no SP cells were detected when aliquots from the same positive specimens, consisting of previously stained 55 x 10(6) cells and fractionated into independent aliquots with 5 x 10(6) cells were used., Conclusions: In this study, we show that there is great variability in SP cell numbers when aliquots obtained either from leukapheresis or bone marrow products represent about 1% of the total product volume. In contrast, when aliquots represented about 12% of the total product volume SP cells measurements were consistent. The high cell number of some specimens can be a limitation for the accurate identification and isolation of the SP compartment. Aliquots containing a minimum of 55 × 10(6) cells should be used for statistical significance.

The aim of supportive autografting is to reduce the side effects from stem cell transplantation and avoid procedure-related health disadvantages for patients at the lowest possible cost and resource expenditure. Economic evaluation of health care is becoming increasingly important. We report clinical and laboratory data collected from 397 consecutive adult patients (173 non-Hodgkin lymphoma, 30 Hodgkin lymphoma, 160 multiple myeloma, 7 autoimmune diseases, and 28 acute leukemia) who underwent their first autologous peripheral blood stem cell transplantation (PBSCT). We considered primary endpoints evaluating health economic efficacy (eg, antibiotic administration, transfusion of blood components, and time in hospital), secondary endpoints evaluating toxicity (in accordance with Common Toxicity Criteria), and tertiary endpoints evaluating safety (ie, the risk of regimen-related death or disease progression within the first year after PBSCT). A time-dependent grading of efficacy is proposed with day 21 for multiple myeloma and day 25 for the other disease categories (depending on the length of the conditioning regimen) as the acceptable maximum time in hospital, which together with antibiotics, antifungal, or transfusion therapy delineates four groups: favorable (≤7 days on antibiotics and no transfusions; ≤21 [25] days in hospital), intermediate (from 7 to 10 days on antibiotics and <3 transfusions, ≤21 to 25 days in hospital or ≥7 days on antibiotics and no transfusions; from 21 to 30 days [25 to 34] in hospital), unfavorable (>7 days on antibiotics, >3 but <6 transfusions; >30/34 days in hospital after transplantation), and very unfavorable (>10 days on antibiotics, >6 transfusions; >30 to 34 days in hospital). The multivariate analysis showed that (1) PBSC harvests of ≥4 × 10(6)/kg CD34 + cells in 1 apheresis procedure were associated with a favorable outcome in all patient categories except acute myelogenous leukemia and acute lymphoblastic leukemia (P = .001), (2) ≥5 × 10(6)/kg CD34 + cells infused predicted better transplantation outcome in all patient categories (P < .0001) except acute myelogenous leukemia and acute lymphoblastic leukemia, (3) 1 or 2 aphereses (P = .001) predicted good outcome, (4) toxicity increased with higher graft volume reinfused (>500 mL) (P = .002), and (5) patients with a central venous catheter during both collection and infusion of PBSC had a more favorable outcome post-PBSCT than peripheral access (P = .007). The type of mobilization regimen did not affect the outcome of auto-PBSCT. The present study identified predictive variables, which may be useful in future individual pretransplantation probability evaluations with the goal to improve supportive care., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: In the absence of a gold standard for hematopoietic progenitor counting, the intra-laboratory variation between commonly used strategies for progenitor assessment was compared., Methods and Materials: We used a pool of FITC-conjugated monoclonal antibodies (CD14, CD11b) and PE-CD34 to facilitate CD34 counting, excluding CD14(+)/CD11b(+) bright cells. We compared this protocol with other common methodologies, such as the single-staining approach, known as the Milan method, and the ISHAGE multiparameter method., Results: We show that the CD14/CD11b protocol is a valid approach to progenitor cell counting. Though different methods give different results for progenitor cell counting, Lin's coefficient shows high concordance among flow cytometry counts but a substantial difference with colony- forming unit counts. Both the ISHAGE and CD14/CD11b protocol give higher counts than the Milan method. Moreover, on revising the ISHAGE analysis, we described a rare population of cells with the CD34(+)CD45(neg) phenotype, which could have an impact in CD34 counting., Conclusions: We have designed a valid alternative approach for hematopoietic progenitor cell counting, and we show that different methods give different results.

The side population (SP) has become an important hallmark for the definition of the stem-cell compartment, especially for the detection of stem cells and for their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34(-) and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, the protocol is difficult for most investigators to perform: first, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because analysis of the acquired data requires extensive expertise in flow cytometry to accurately detect the SP events. More recently, a new assay based on the efflux of Vybrant DyeCycle Violet stain (DCV) has been documented to discriminate SP cells. This unit contains many helpful pointers to aid the user in obtaining the best possible results with these assays., (© 2013 by John Wiley & Sons, Inc.)

Due to their favorable intrinsic features, including engraftment, differentiation, and immunomodulatory potential, adult mesenchymal stem cells (MSCs) have been proposed for therapeutic in utero intervention. Further improvement of such attributes for particular diseases might merely be achieved by ex vivo MSC genetic engineering previous to transplantation. Here, we evaluated for the first time the feasibility, biodistribution, long-term engraftment, and transgenic enhanced green fluorescent protein (EGFP) expression of genetically engineered human adipose tissue-derived MSCs (EGFP(+)-ASCs) after intra-amniotic xenotransplantation at E17 of gestation into our validated pregnant rabbit model. Overall, the procedure was safe (86.4% survival rate; absence of anatomical defects). Stable, low-level engraftment of EGFP(+)-ASCs was confirmed by assessing the presence of the pWT-EGFP lentiviral provirus in the young transplanted rabbit tissues. Accordingly, similar frequencies of provirus-positive animals were found at both 8 weeks (60%) and 16 weeks (66.7%) after in utero intervention. The presence of EGFP(+)-ASCs was more frequent in respiratory epithelia (lung and trachea), according to the route of administration. However, we were unable to detect EGFP expression, neither by real-time polymerase chain reaction nor by immunohistochemistry, in the provirus-positive tissues, suggesting EGFP transgene silencing mediated by epigenetic events. Moreover, we noticed lack of both host cellular immune responses against xenogeneic ASCs and humoral immune responses against transgenic EGFP. Therefore, the fetal microchimerism achieved by the EGFP(+)-ASCs in the young rabbit hosts indicates induction of donor-specific tolerance after fetal rabbit xenotransplantation, which should boost postnatal transplantation for the early treatment/prevention of many devastating congenital disorders.

Prenatal transplantation of genetically engineered mesenchymal stem cells (MSCs) might benefit prevention or treatment of early-onset genetic disorders due to the cells' intrinsic regenerative potential plus the acquired advantage from therapeutic transgene expression. However, a thorough assessment of the safety, accessibility, and behavior of these MSCs in the fetal environment using appropriate animal models is required before we can advance toward a clinical application. We have recently shown that fetal rabbit liver MSCs (fl-MSCs) have superior growth rate, clonogenic capability, and in vitro adherence and differentiation abilities compared with adult rabbit bone marrow MSCs. In this follow-up study, we report safe and widespread distribution of recombinant pSF-EGFP retrovirus-transduced fl-MSCs (EGFP(+)-fl-MSCs) in neonatal rabbit tissues at 10 days after fetal allogeneic transplantation through both intrahepatic and intra-amniotic administration. Conversely, a more restricted biodistribution pattern according to the route of administration was apparent in the young rabbits intervened at 16 weeks after fetal EGFP(+)-fl-MSC transplantation. Furthermore, the presence of these cells in the recipients' tissues, tracked with the reporter provirus, was inversely related to the developmental stage of the fetuses at the time of intervention. Long-term engraftment was confirmed both by fluorescence in situ hybridization analysis on touch tissue imprints using a chromosome Y-specific BAC probe, and by immunohistochemical localization of EGFP expression. Finally, there was no evidence of immune responses against the transplanted EGFP(+)-fl-MSCs or the EGFP transgenic product in the treated young rabbits. Thus, cell transplantation approaches using genetically engineered fetal MSCs may prove particularly valuable to frontier medical treatments for congenital birth defects in perinatology.

The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents., (Copyright © 2011 International Society for Advancement of Cytometry.)

Because of their abundance and ease of isolation, multilineage differentiation, and paracrine and immunoregulatory capabilities, genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) might combine cell- and gene therapy-based strategies for efficacious tissue repair/regeneration. In this report, we aimed to analyze and influence the long-term dynamics of transgene expression in ASCs transduced with different gammaretroviral vector configurations incorporating the human β-interferon scaffold attachment region (IFN-SAR) and/or chicken 5'HS4 β-globin insulator sequences. In our undifferentiated ASC culture model, naked retroviral vectors experienced EGFP transgene extinction correlating with increases in both H3 histone deacetylation and CpG dinucleotide methylation within the 5' long terminal repeat-primer-binding site proviral region. Retroviral configurations incorporating the referred boundary elements alone or combined were able to prevent the development of the above epigenetic events and to reduce transgene extinction to different degrees. Particularly, the IFN-SAR sustained the highest levels of H3 histone acetylation and transgene expression throughout the study. Analogously, ASCs differentiating to adipocytes or osteocytes experienced a gradual decline of EGFP expression using naked retroviral vectors. In contrast, only retroviral configurations including the IFN-SAR alone were able to overcome the epigenetic pressure, yielding high-level, uniform transgene expression throughout both lineage differentiation processes. Thus, embedding the IFN-SAR in retroviral vectors should have positive implications in gene repair strategies using ASCs.

Objective: 1) To evaluate whether peripheral blood mononuclear cells (PBMCs) from type 2 diabetic patients present an impairment of phagocytic activity; 2) To determine whether the eventual impairment in phagocytic activity is related to glycemic control and can be reversed by improving blood glucose levels., Methods: 21 type 2 diabetic patients and 21 healthy volunteers were prospectively recruited for a case-control study. In addition, those patients in whom HbA1c was higher than 8% (n = 12) were hospitalized in order to complete a 5-day intensification treatment of blood glucose. Phagocytic activity was assessed by using a modified flow cytometry procedure developed in our laboratory based on DNA/RNA viable staining to discriminate erythrocytes and debris. This method is simple, highly sensitive and reproducible and it takes advantage of classic methods that are widely used in flow cytometry., Results: Type 2 diabetic patients showed a lower percentage of activated macrophages in comparison with non-diabetic subjects (54.00±18.93 vs 68.53±12.77%; p = 0.006) Significant negative correlations between phagocytic activity and fasting glucose (r = -0.619, p = 0.004) and HbA1c (r = -0.506, p = 0.019) were detected. In addition, multiple linear regression analyses showed that either fasting plasma glucose or HbA1c were independently associated with phagocytic activity. Furthermore, in the subset of patients who underwent metabolic optimization a significant increase in phagocytic activity was observed (p = 0.029)., Conclusions: Glycemic control is related to phagocytic activity in type 2 diabetes. Our results suggest that improvement in phagocytic activity can be added to the beneficial effects of metabolic optimization.

Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-beta SAR and/or 5'HS4 beta-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability. Conversely, the SAR/5'HS4 insulator combination appeared to increase the homogeneity of EGFP expression in mass cultures. Furthermore, a clonal analysis of the dispersion of EGFP expression revealed that the IFN-SAR/5'HS4 insulator dyad was particularly effective in reducing the variability of transgene expression when both sequences were placed in opposite orientations within the retroviral backbone. These results may prove useful for the design of more stable retroviral expression cassettes able to counteract chromosomal position effects.

Numerous studies conducted in a diversity of adult tissues have shown that certain stem cells are characterized by the expression of a protein known as the ABCG2 transporter (where ABC is ATP- binding cassette). In the adult pancreas, although various multipotent progenitors have been proposed, the ABCG2 marker has only been detected in the so-called 'side population' (a primitive haematopoietic cell population with a multipotential capacity). In the present study we sought to identify new ABCG2+ pancreatic cell populations and to explore whether they exhibit the properties of progenitor cells. We isolated and expanded mitoxantrone-resistant cells from pancreata of lactating rats by drug selection. These cells were characterized and maintained in different stages of differentiation using several media 'cocktails' plus Matrigel (BD Biosciences). Differentiation was assessed by RT-PCR (reverse transcription-PCR), immunocytochemistry, electron microscopy and ELISA. The expanded cell population demonstrated a phenotype of PaSCs (pancreatic stellate cells). Spontaneous cell clusters occurred during cell expansion and they showed weak expression of the transcription factor Pdx1 (pancreatic and duodenal homeobox 1). Moreover, the presence of inductive factors in the Matrigel plus exendin-4 led to an increase in Pdx1 and endocrine genes, such as insulin, islet amyloid polypeptide, glucagon, the glucose transporter GLUT2, chromogranin A and the convertases PC1/3 and PC2 were also detected. Immunocytochemical analysis showed co-localization of insulin and C-peptide, whereas ultrastructural studies revealed the presence of granules. Insulin secretion from cell clusters was detected in the cell culture medium. We identified a population of PaSCs that express the ABCG2+ transporter and have the capacity to transdifferentiate into insulin-producing cells. Although the potential therapeutic application remains to be tested, PaSCs could represent a future option for insulin replacement in diabetes research.

P-glycoprotein (Pgp), a protein codified by Multi Drug Resistance (MDR1) gene, has a detoxifying function and might influence the toxicity and pharmacokinetics and pharmacodynamics of drugs. Sampling strategies to improve Pgp studies could be useful to optimize the sensitivity and the reproducibility of efflux assays. This study aimed to compare Pgp expression and efflux activity by measuring Rhodamine123 (Rh123) retention in lymphocytes stored under different conditions, in order to evaluate the potential utility of any of the storing conditions in Pgp functionality. Our results show no change in protein expression of Pgp by confocal studies and Western blotting, nor changes at the mRNA level (qRT-PCR). No differences in Rh123 efflux by Pgp activity assays were found between fresh and frozen lymphocytes after 24 hours of blood extraction, using either of the two Pgp specific inhibitors (VP and PSC833). Different working conditions in the 24 hours post blood extraction do not affect Rh123 efflux. These results allow standardization of Pgp activity measurement in different individuals with different timing of blood sampling and in different geographic areas.

Background: Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation., Methods: To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose-response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated., Results: With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not., Conclusions: This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation.

There is accumulating evidence that omega-3 fatty acids may modulate immune responses. When monocytes were differentiated to dendritic cells (DCs) in the presence of docosahexaenoic acid (DHA), the expression of costimulatory and antigen presentation markers was altered in a concentration-dependent way, positively or negatively, depending on the markers tested and the maturation stage of the DCs. Changes induced by eicosapentaenoic acid and linoleic acid were similar but less intense than those of DHA, whereas oleic acid had almost no effect. DHA-treated, mature DCs showed inhibition of IL-6 expression and IL-10 and IL-12 secretion, and their lymphoproliferative stimulation capacity was impaired. The phenotypic alterations of DCs induced by DHA were similar to those already reported for Rosiglitazone (Rosi), a peroxisome proliferator-activated receptor gamma (PPAR gamma) activator, and the retinoid 9-cis-retinoic acid (9cRA), a retinoid X receptor (RXR) activator. Moreover, DHA induced the expression of PPAR gamma target genes pyruvate dehydrogenase kinase-4 and aP-2 in immature DCs. The combination of DHA with Rosi or 9cRA produced additive effects. Furthermore, when DCs were cultured in the presence of a specific PPAR gamma inhibitor, all of the changes induced by DHA were blocked. Together, these results strongly suggest that the PPAR gamma:RXR heterodimer is the pathway component activated by DHA to induce its immunomodulatory effect on DCs.

Background: Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors., Methods: Rabbit fetuses received 8.5 x 10(6) HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post-in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein., Results: Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP., Conclusions: At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer., ((c) 2008 John Wiley & Sons, Ltd.)

Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERbeta) remain elusive., Methods: We have analyzed the levels of ERbeta1 and ERbeta2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERbeta1 in the human prostate cancer LNCaP cell line., Results: Both ERbeta1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERbeta2 levels decreased during the S phase and increased in the G2/M phase. ERbeta1 protein was detected in both the nuclear and non-nuclear fractions, and ERbeta2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERbeta was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFkappaB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERbeta1 or ERbeta1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERbeta1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1-ERbeta1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested., Conclusions: Our results demonstrate that, in LNCaP prostate cancer cells, both ERbeta isoforms are differentially expressed during the cell cycle and that ERbeta regulates the G1 phase by a non-genomic mechanism.

At nanomolar range, 9-cis-retinoic acid (9cRA) was able to interfere in the normal differentiation process from human monocyte to immature dendritic cell (DC) and produced a switch in mature DCs to a less stimulatory mode than untreated cells. 9cRA-treated mature DCs secreted high levels of IL-10 with an IL-12 reduced production. The phenotypic alterations unleashed by 9cRA were similar but not identical to other specific retinoid X receptor (RXR) agonists and to those already reported for rosiglitazone, a PPARgamma activator, on DCs. The simultaneous addition of 9cRA and rosiglitazone on DCs displayed additive effects. Moreover, addition to cultures of GW9662, a specific inhibitor of PPARgamma, or the RXR pan-antagonist HX603, blocked these changes. All these results suggest an activation of PPARgamma-RXR and other RXR containing dimers by 9cRA in DCs. Finally, both GW9662 and HX603 by themselves altered the maturation process unleashed by TNFalpha, poly(I:C) or LPS on human DCs further suggesting that the heterodimer PPARgamma-RXR must fulfill a significant role in the physiological maturation process of these cells in addition to the repressing effects reported till now for this nuclear receptor.