Carboxyfluorescein Diacetate Succinimidyl Ester Labeling Research Articles

The Effect and Mechanism of Astragalus Polysaccharides on T Cells and Macrophages in Inhibiting Prostate Cancer

BackgroundThe impact and underlying mechanisms of astragalus polysaccharide (APS) on prostate cancer, particularly its role in immunomodulation, remain inadequately elucidated. MethodsThis study employed the XTT assay for assessing proliferation in prostate cancer cells and macrophages. T cell proliferation was determined using the Carboxyfluorescein diacetate succinimidyl ester labeling assay. APS's effect on T cells and macrophages was scrutinized via flow cytometry, Western blot analysis, ELISA, quantitative PCR and cytokine membrane arrays. The effect of APS on interaction between PD-L1 and PD-1 was investigated by the PD-L1/PD-1 homogeneous assay. Additionally, the impact of conditioned medium from T cells and macrophages on PC-3 cell migration was explored through migration assays. ResultsIt was observed that APS at concentrations of 1 and 5 mg/mL enhanced the proliferation of CD8+ T cells. At a concentration of 5 mg/mL, APS activated both CD4+ and CD8+ T cells, attenuated PD-L1 expression in prostate cancer cells stimulated with interferon gamma (IFN-γ) or oxaliplatin, and moderately decreased the population of PD-1+ CD4+ and PD-1+ CD8+ T cells. Furthermore, APS at this concentration impeded the interaction between PD-L1 and PD-1, inhibited the promotion of prostate cancer migration mediated by RAW 264.7 cells, THP-1 cells, CD4+ T cells, and CD8+ T cells, and initiated apoptosis in prostate cancer cells treated with conditioned medium from APS (5 mg/mL)-treated CD8+ T cells, RAW 264.7 cells, or THP-1 cells. ConclusionThe findings indicate a potential role of 5 mg/mL APS in modulating the PD-1/PD-L1 pathway and influencing the immune response, encompassing T cells and macrophages. Consequently, further in vivo research is recommended to assess the efficacy of APS.

Effects of interleukin 6 and tumor necrosis factor-α on the proliferation of porcine theca interna cells: Possible role of these cytokines in the pathogenesis of polycystic ovary syndrome

ObjectiveWe studied the effects of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) on the proliferation of porcine theca interna (TI) cells and further elucidated the roles of IL-6 and TNF-α in the pathogenesis of polycystic ovary syndrome. Materials and MethodsTI cells were treated with 10 pg/mL, 100 pg/mL, and 1000 pg/mL IL-6 or TNF-α. TI cell proliferation was then examined by carboxyfluorescein diacetate succinimidyl ester labeling and flow cytometry. ResultsCell proliferation was not significantly different in TI cells cultured in medium alone (control) or in the presence of IL-6. At 72 hours of treatment, the mean fluorescence intensity was significantly lower in TI cells treated with 100 pg/mL and 1000 pg/mL TNF-α than in the control (p < 0.05). ConclusionTNF-α, but not IL-6, was able to promote TI cell proliferation. Our results suggest that TNF-α might play a role in hyperandrogenism, cortex thickness, and the increased ovary volume observed in polycystic ovaries.

Abstract B48: Intratumor ploidy heterogeneity as a chemoresistance mechanism in triple-negative breastcancer

Abstract Background: Triple negative breast cancers (TNBC) are aggressive, heterogeneous tumors, and currently lack targeted therapies. Chemotherapy is the mainstay treatment, but tumor recurrence is a major cause of mortality and morbidity. Chemotherapy may select for resistant subclones that have a survival advantage over the bulk population of cancer cells and cause tumor relapse. DNA aneuploidy (abnormal number of chromosomes) is a common genetic feature in breast cancer and has been frequently associated with poor prognosis. Although, aneuploid or polyploid cancer cells can contribute to intratumor ploidy heterogeneity, their functional role in tumorigenesis and chemoresistance is largely undefined. Traditionally, polyploid cells were thought to arise due to defects in cell division and were considered to be senescent. However, recent reports have shown that polyploid cells represent a viable and proliferating subpopulation within a tumor and may have a direct role in regulating chemoresistance. Methods: In this study, we isolated and functionally characterized the diploid and polyploid sub-populations derived from TNBC cell lines. DNA ploidy was assessed in Hoechst 33342 stained cells using flow cytometry. Cells with greater than 4N DNA content were considered as polyploid. Next, we analyzed cell proliferation in HCC1937 cells using CFSE and DNA labeling. Live-cell imaging was performed on flow-sorted diploid and polyploid cells using IncuCyte Zoom to monitor cell proliferation in the absence and presence of mitotic inhibitor (docetaxel). Results: DNA ploidy analysis identified presence of polyploid cells in HCC1395 and HCC1937 cell lines at a frequency of 6.7% and 5.8% respectively. Polyploid cells have higher forward-scatter than diploid cells suggesting that they are larger in size. In addition, nuclear staining with Hoechst 33342 showed that giant polyploid cells contain enlarged and/or multiple nuclei. Time-lapse microscopy in polyploid cells revealed mitotic structures suggestive of multipolar cell division. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeling showed that polyploid cycle slower than the predominant diploid population. Further, the IncuCyte cell proliferation assay also confirmed that sorted polyploid cells divide slower than the diploid cells and displayed resistance to docetaxel. Concusions: These findings indicate that there are functional differences between diploid and polyploid sub-populations. In our future studies, we will examine the isolated diploid and polyploid cells for differences in their tumorigenic potential and role in tumor recurrence. Citation Format: Rekha Gyanchandani, Adrian Lee. Intratumor ploidy heterogeneity as a chemoresistance mechanism in triple-negative breastcancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B48.

Activation of GPR30 inhibits cardiac fibroblast proliferation.

The incidence of left ventricular diastolic dysfunction significantly increases in postmenopausal women suggesting the association between estrogen loss and diastolic dysfunction. The in vivo activation of G protein-coupled estrogen receptor (GPR30) attenuates the adverse effects of estrogen loss on cardiac fibrosis and diastolic dysfunction in mRen2.Lewis rats. This study was designed to address the effects of GPR30 on cardiac fibroblast proliferation in rats. The expression of GPR30 in cardiac fibroblasts isolated from adult Sprague-Dawley rats was confirmed by RT-PCR, Western blot analysis, and immunofluorescence staining. Results from BrdU incorporation assays, cell counting, carboxyfluorescein diacetate succinimidyl ester labeling in conjunction with flow cytometry, and Ki-67 staining showed that treatment with G1, a specific agonist of GPR30, inhibited cardiac fibroblast proliferation in a dose-dependent manner, which was associated with decreases in CDK1 and cyclin B1 protein expressions. In the GPR30-KO cells, BrdU incorporation, and CDK1 and cyclin B1 expressions significantly increased when compared to GPR30-intact cells. G1 had no effect on BrdU incorporation, CDK1 and cyclin B1 mRNA levels in GPR30-KO cells. In vivo studies showed increases in CDK1 and cyclin B1 mRNA levels, Ki-67-positive cells, and the immunohistochemistry staining of vimentin, a fibroblast marker, in the left ventricles from ovariectomized mRen2.Lewis rats versus hearts from ovary-intact littermates; 2 weeks of G1 treatment attenuated these adverse effects of estrogen loss. This study demonstrates that GPR30 is expressed in rat cardiac fibroblasts, and activation of GPR30 limits proliferation of these cells likely via suppression of the cell cycle proteins, cyclin B1, and CDK1.

Carboxyfluorescein diacetate succinimidyl ester labeling method to study the interaction between Leptospira and macrophages

Leptospirosis, which is caused by pathogenic species of the genus Leptospira, has emerged as one of the most widespread zoonotic diseases in the world. The exact mechanism of pathogenesis remains unknown, and the interaction between Leptospira and macrophages is not well understood. In this study, we report that carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) can efficiently label different Leptospira interrogans strains without affecting bacterial motility, viability, or virulence. Following co-incubation, CFDA-SE-labeled leptospires associated with macrophages were quantified by flow cytometry or confocal microscopy. In addition, we showed that trypan blue efficiently quenched the extracellular fluorescence from the adherent leptospires, which enabled intracellular and extracellular bacteria to be distinguished.

Dry eye-induced CCR7+CD11b+ cell lymph node homing is induced by COX-2 activities.

We aimed to determine the role of CCR7+CD11b+ cell lymph node (LN) homing and T-cell differentiation in dry eye (DE)-induced immunopathogenesis and investigate the therapeutic effects of cyclooxygenase-2 (COX-2) and prostaglandin E2/eicosanoid-prostanoid (PGE2/EP) inhibitors against DE. Six-week-old female C57BL/6 mice were housed in a controlled-environment chamber and administered topical selective COX-2 inhibitors or EP2 antagonists. Expression of major histocompatibility complex (MHC)-IIhigh, CD11b+, CCR7+, IFN-γ+, IL-17+, and CD4+ in the corneas and draining LNs was evaluated using flow cytometry. Mixed lymphocyte reactions (MLRs) with carboxyfluorescein diacetate succinimidyl ester labeling and intracellular cytokine staining were used to verify DE-induced corneal dendritic cell function. mRNA expression of COX-2, EPs, and proinflammatory cytokines in ocular surface was evaluated using quantitative RT-PCR and immunohistochemical staining. Dry eye significantly increased MHC-IIhighCD11b+ and CCR7+CD11b+ cells in the cornea and LNs, and MLR revealed CCR7+CD11b+ cells from DE corneas stimulated IL-17+CD4+ cell proliferation. mRNA levels of COX-2, EP2, IFN-γ, TNF-α, IL-6, and IL-17 were significantly higher in DE ocular surface but were suppressed by topical COX-2 inhibitors and EP2-specific blockers. Immunohistochemical staining showed COX-2 and matrix metalloproteinase expression in DE corneal epithelia that was diminished by both topical treatments. Furthermore, both topical treatments significantly reduced frequencies of MHC-IIhigh, CD11b+, and CCR7+CD11b+ cells in the corneas and LNs, but also IL-17+CD4+ cells in LNs. Topical COX-2/EP2 treatment reduces CCR7+CD11b+ cells on the ocular surface with inhibition of cellular LN homing and suppresses Th17 immune response, suggesting the COX-2/PGE2/EP axis contributes to immuno-inflammatory pathogenesis on the ocular surface and may be a novel therapeutic target in DE.

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

Optimization of Immunosuppressive Therapy Based on a Multiparametric Mixed Lymphocyte Reaction Assay Reduces Infectious Complications and Mortality in Living Donor Liver Transplant Recipients

AimWe investigated the clinical relevance of immune monitoring by a multiparametric mixed lymphocyte reaction (MLR) assay, wherein the number and phenotype of alloreactive precursors can be quantified by combining the results of carboxyfluorescein diacetate succinimidyl ester labeling and flow cytometry analysis. MethodsIn 51 adult patients undergoing living donor liver transplantation (OLT), immunosuppressive drugs were dosed on the basis of immune monitoring by the MLR assay (optimized protocol: group O). In 64 other patients, the agents were prescribed according to empirical regimens (empirical protocol: group E). In group O, MLR assays were performed at 2- to 4-week intervals until 3 months after OLT and thereafter at 3- to 6-month intervals. Therapeutic adjustments for immunosuppressants were determined by tapering the doses in cases of anti-donor hyporesponsiveness for both CD4+ and CD8+ T-cell subsets. ResultsThe 1-year patient and graft survivals in groups O versus E were 90.2% versus 76.6%, respectively. The incidence of acute rejection episodes (ARE) among group O (13.7%) were lower than in cohort E (28.1%). None of the patients in group O while four patients (3%) in group E already have shown chronic rejection to date. The incidences of bacteremia and fungal infections in group O (9.8% and 7.5%, respectively) were lower than in cohort E (18.8% and 12.6%, respectively). ConclusionA multiparametric MLR assay may facilitate the development of adequate immunosuppressive regimens.

A novel fluorescent-based assay reveals that thrombopoietin signaling and Bcl-X L influence, respectively, platelet and erythrocyte lifespans

The factors determining platelet and erythrocyte lifespan are not completely understood, despite extensive study. The lack of success may be attributed to the methods used to measure lifespan kinetics, all of which require processing of cells prior to analysis, and the inconsistent and potentially inappropriate use of mathematical models for data analysis. The aims of this study were to establish an in vivo platelet and erythrocyte labeling method using carboxyfluorescein diacetate succinimidyl ester (CFSE), determine the most appropriate mathematical model for lifespan analysis, and apply both to the study of factors that control platelet and erythrocyte lifespans. Control, c-mpl knockout (KO), and Bcl-X(L) mutant mice were injected with CFSE and platelet and erythrocyte fluorescence followed over time. Datasets were analyzed using linear, exponential, multiple-hit, and lognormal mathematical models. In vivo CFSE labeling of platelets and erythrocytes requires no postcollection processing, proved stable, nontoxic, nonimmunogenic, and the lifespans were highly reproducible. Mathematical modeling revealed the lognormal model gave a robust fit to control and extreme datasets when either extrinsic or intrinsic factors determined lifespan. Using these methods, platelet lifespans were found to be significantly shortened in thrombopoietin-receptor-deficient mice independent of blood loss, and the antiapoptotic protein Bcl-X(L) was shown to play a role in prolonging erythrocyte lifespans. The simultaneous study of platelet and erythrocyte lifespans using in vivo CFSE labeling with lognormal modeling yielded insight into common intrinsic and extrinsic platelet and erythrocyte lifespan determinants and provides an improved methodology for use in this field of research.

Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling

Peritoneal B cells represent a heterogeneous mixture of mature peripheral B lineage subsets with distinct developmental and functional characteristics. Here, we report that a single intraperitoneal injection of intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) results in the stable fluorescent labeling of resident lymphocytes, without dissipation of the tracer compound into other peripheral lymphoid organs. Using this in situ labeling procedure followed by multicolor flow cytometry or tissue fluorescence at various periods for up to 4 weeks post-labeling, we demonstrate that the distinct peritoneal leukocyte sub-populations and, within the B lineage, B-1 and B-2 B-cell subsets have different exchange kinetics with extraperitoneal sites under steady-state conditions. The B cells labeled with CFSE showed only minimal localization to other peripheral lymphoid tissues. On the other hand, a substantial fraction of both B-1 and B-2 subsets labeled with CFSE accumulated within the pleural cavity, although at a lower frequency than in the peritoneum. We also show that exposure to LPS induces a rapid re-distribution of peritoneal B lymphocytes and an enhanced entry of B-1 cells in the pleural cavity, in addition to augmenting the egress and the division-linked reduction of CFSE fluorescence of both B-1 and B-2 cells. These data indicate that following their in situ labeling, peritoneal lymphocytes show preferential accumulation in serosal cavities, although with a differential efficiencies for T, B-1 and B-2 lymphocyte subsets.

Regulation of Rat and Human T-Cell Immune Response by Pharmacologically Modified Dendritic Cells

The central function of dendritic cells (DC) in inducing and preventing immune responses makes them ideal therapeutic targets for the induction of immunologic tolerance. In a rat in vivo model, we showed that dexamethasone-treated DC (Dex-DC) induced indirect pathway-mediated regulation and that CD4+CD25+ T cells were involved in the observed effects. The aim of the present study was to investigate the mechanisms underlying the acquired immunoregulatory properties of Dex-DC in the rat and human experimental systems. After treatment with dexamethasone (Dex), the immunogenicity of Dex-DC was analyzed in T-cell proliferation and two-step hyporesponsiveness induction assays. After carboxyfluorescein diacetate succinimidyl ester labeling, CD4+CD25+ regulatory T-cell expansion was analyzed by flow cytometry, and cytokine secretion was measured by ELISA. In this study, we demonstrate in vitro that rat Dex-DC induced selective expansion of CD4+CD25+ regulatory T cells, which were responsible for alloantigen-specific hyporesponsiveness. The induction of regulatory T-cell division by rat Dex-DC was due to secretion of interleukin (IL)-2 by DC. Similarly, in human studies, monocyte-derived Dex-DC were also poorly immunogenic, were able to induce T-cell anergy in vitro, and expand a population of T cells with regulatory functions. This was accompanied by a change in the cytokine profile in DC and T cells in favor of IL-10. These data suggest that Dex-DC induced tolerance by different mechanisms in the two systems studied. Both rat and human Dex-DC were able to induce and expand regulatory T cells, which occurred in an IL-2 dependent manner in the rat system.

Proliferation and differentiation of oligodendrocyte progenitor cells induced from rat embryonic neural precursor cells followed by flow cytometry

Previous studies have shown that a cell-intrinsic timer might determine when oligodendrocyte progenitor cells (OPCs) isolated from the central nervous system (CNS) stop dividing and initiate differentiation in a defined environment. In this report, the proliferation and differentiation of OPCs induced from neural precursor cells (NPCs) were analyzed by flow cytometry combined with carboxyfluorescein diacetate succinimidyl ester labeling and propidium iodide staining, respectively. When OPCs were cultured in OPC-medium, more than 30% of cells were in S- and G2/M-phases, and continuously self-renewed without differentiation. After exposure to thyroid hormone, there was an obvious decrease in the fraction of cells in both S- and G2/M-phases (<10%). Furthermore, the OPCs no longer proliferated, but differentiated into oligodendrocytes. The dynamic proliferation and differentiation characteristics of OPCs induced from NPCs and analyzed by flow cytometry were similar to those of OPCs isolated from the CNS and analyzed by other methods. These studies indicated that the proliferation and differentiation of OPCs can be followed simply and rapidly by flow cytometry.

Fluorescent Eimeria bovis sporozoites and meront stages in vitro: a helpful tool to study parasite–host cell interactions

A fluorescence-based technique was established to trace intracellular sporozoites of Eimeria bovis for tests on gliding motility, invasion, replication and quantification of infection rates in cultured bovine umbilical vein endothelial cells (BUVEC) by laser scanning confocal microscopy and flow cytometry (FCM) analyses. Employing the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), we determined its effects on sporozoites at various concentrations and duration of staining. More than 98% of sporozoites were labelled with the dye at a concentration of 2.5 muM. Staining was predominantly found in refractile bodies and presumptive micronemes. Upon infection of BUVEC, CFSE-labelled sporozoites developed into fluorescent immature macromeronts, which were traceable inside the cells until 22 days postinfection (p. i.). Consistent with a peripheral localisation of the fluorescence signal in macromeronts merozoites released from these lacked detectable fluorescence. As example of use, a multicolour FCM approach for the simultaneous determination of E. bovis infection and host cell surface molecule expression was established. The approach proved suitable to quantify major histocompatibility complex (MHC-I) and MHC-II expression, thereby clearly distinguishing between infected and uninfected BUVEC up to day 14 p. i. In conclusion, CFSE labelling of E. bovis sporozoites facilitates monitoring of intracellular stages in vitro and will be a highly useful tool for studying host cell responses towards parasite invasion.

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.

Novel Model of Antigen-Specific Induction of Bile Duct Injury

Biliary-directed inflammation is an important cause of acute and chronic liver disease. We developed and characterized a transgenic mouse model of immune-mediated hepatobiliary injury. Ovalbumin (OVA)-BIL mice were developed using 3.0 kilobase of the rat apical sodium-dependent bile acid transporter promoter to drive aberrant expression of a membrane form of ovalbumin (OVA) on biliary epithelium. Liver inflammation resulted from adoptive transfer of OVA-specific T cells. Liver immune cells were characterized to determine the mechanism of the response by assessing activation, proliferation, and intracellular cytokine expression. OVA-BIL transgenic mice were tolerant to OVA, without evidence of liver disease. Adoptive transfer of OVA-specific CD4+ and CD8+ T cells into naïve OVA-BIL mice led to biliary-centered necroinflammatory damage in a dose-dependent manner. This inflammation absolutely required CD8+ T cells and was augmented by CD4+ T cells. Adoptively transferred OVA CD8+ cells homed to and proliferated in the liver but not the spleen. These activated, adoptively transferred cytotoxic T lymphocytes produced elevated levels of tumor necrosis factor alpha and interferon gamma. T-cell recognition of antigen aberrantly expressed on bile duct epithelium induced an acute necroinflammatory response specific to the liver, with activation, proliferation, and cytokine production predominantly by the OVA-specific cytotoxic T cells. Thus, OVA BIL represents an antigen-specific animal model of inflammatory bile duct injury.

Peripheral Blood B-Cell Death Compensates for Excessive Proliferation in Lymphoid Tissues and Maintains Homeostasis in Bovine Leukemia Virus-InfectedSheep

The size of a lymphocyte population is primarily determined by a dynamic equilibrium between cell proliferation and death. Hence, lymphocyte recirculation between the peripheral blood and lymphoid tissues is a key determinant in the maintenance of cell homeostasis. Insights into these mechanisms can be gathered from large-animal models, where lymphatic cannulation from individual lymph nodes is possible. In this study, we assessed in vivo lymphocyte trafficking in bovine leukemia virus (BLV)-infected sheep. With a carboxyfluorescein diacetate succinimidyl ester labeling technique, we demonstrate that the dynamics of lymphocyte recirculation is unaltered but that accelerated proliferation in the lymphoid tissues is compensated for by increased death in the peripheral blood cell population. Lymphocyte homeostasis is thus maintained by biphasic kinetics in two distinct tissues, emphasizing a very dynamic process during BLV infection.

Detection and properties of the human proliferative monocyte subpopulation

Peripheral blood monocyte subpopulations have been reported and can give rise to diverse, differentiated phenotypes. A subpopulation(s) of human monocytes can proliferate in vitro in response to macrophage-colony stimulating factor (M-CSF; or CSF-1). This population, termed the proliferative monocyte (PM), is presumably less mature than other monocytes; however, it has not been defined further. Previous studies monitoring the frequency of the slowly cycling PM from different donors indicated that the assay for their reproducible measurement required improvement. We demonstrate that for optimal PM detection, high 5-bromo-2'-deoxyuridine concentrations are required over a delayed and wide time-frame. Surface marker phenotyping by flow cytometry showed that freshly isolated PM are CD14+ and could be distinguished from two other human monocyte subpopulations, namely, the CD14lo CD16+ and CD14lo CD64- subsets. PM express relatively high levels of CD64 and CD33 but have relatively low CD13 expression; they are also c-Fms+ and human leukocyte antigen-DR+. Labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) enabled the estimation of the number of PM divisions over time. Following CFSE labeling and culture, PM were sorted from the nonproliferating population and shown to have a distinctive, spindle-shaped morphology and higher capacity to form multinucleated, tartrate-resistant acid phosphatase+ cells in the presence of M-CSF and receptor activator of nuclear factor-kappaB ligand. The phenotype and properties of the PM subpopulation were examined as a prelude to determining its role in disease using methods that can be applied to clarify human monocyte heterogeneity.

Differential Requirement for Cell Division in the Development of Terminally Differentiated Neutrophils and Macrophages from Their Common Progenitors.

Neutrophils and monocytes are the main population of peripheral blood nucleated cells and progenies of common progenitor cells, CFU-GM, that are derived from hematopoietic stem cells. Monocytes are non-dividing immediate precursors for macrophages. Common clinical observation that monocytes more rapidly recover than do neutrophils in myelosuppressive patients indicates the difference in regulatory mechanism of terminal differentiation between neutrophils and macrophages. In this study, we investigated the cell division profiles in the differentiation pathway from common progenitors for neutrophils and macrophages to respective post-mitotic cells in culture. We prepared lineage-negative (Lin−) bone marrow cells from normal C57BL/6 mice by using immunomagnetic beads. The Lin− cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), a cytoplasmic dye that is equally diluted between daughter cells, and stained with anti-Sca-1 antibody and lineage antibodies. CFSEhighSca-1+Lin− cells were sorted on a FACSVantage or FACSAria. When the CFSEhighSca-1+Lin− cells were cultured in the presence of steel factor (SF) and IL-11 for 5 days, the resulting cells could be separated into four populations based on staining with anti-fms and anti-Gr-1 antibodies: fms-Gr-1−, fms+Gr-1−, fms−Gr-1+, and fms+Gr-1+ cells. The fms+Gr-1+ cells showed morphological features typical of macrophages. The commitment of fms−Gr-1+ cells into neutrophilic lineage was evident, although they exhibited an immature morphology. CFSE labeling pattern in fms+Gr-1+ macrophages was equal to that of fms−Gr-1+ neutrophilic cells, indicating that fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells passed through similar successive rounds of cell division in culture of CFSEhighSca-1+Lin− cells. These fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells were subsequently cultured for additional 3 days in the presence of SF, IL-11, IL-3, GM-CSF, G-CSF, M-CSF, erythropoietin, and thrombopoietin. While a decline in the number of fms+Gr-1+ macrophages was observed, the number of fms−Gr-1+ neutrophilic cells increased approximately 5-fold during subsequent culture. The proliferation of fms−Gr-1+ neutrophilic cells and no cell division of fms+Gr-1+ macrophages were confirmed by Ki-67 antibody staining, BrdU incorporation, and DNA staining. The cultured fms−Gr-1+ neutrophilic cells also predominantly acquired a mature morphology. These results suggest distinct cell division histories between terminally differentiated neutrophils and macrophages, possibly explaining the early appearance of monocytes in the blood after myeloablative therapy, as compared to neutrophils.

Low Incidence of Acute Rejection after Living-Donor Liver Transplantation: Immunologic Analyses by Mixed Lymphocyte Reaction using a Carboxyfluorescein Diacetate Succinimidyl Ester Labeling Technique

To monitor antidonor alloreactivity for accurate diagnosis of acute rejection after living-donor liver transplantation (LDLT), we used a mixed lymphocyte reaction (MLR) assay using an intracellular fluorescent dye carboxyfluorescein diacetate succimidyl ester (CFSE)-labeling technique (CFSE-MLR) in 29 consecutive patients who underwent adult-to-adult LDLT. For patients who developed moderate or severe disorders in liver function, CFSE-MLR was performed together with needle biopsy of the liver allografts immediately after liver dysfunction had occurred. CFSE-labeled peripheral blood mononuclear cells (PBMC) from recipients and irradiated autologous, donor, or third-party PBMC were cultured, and then proliferation and CD25 expression in each of the CD4+ and CD8+ T cell subsets were analyzed by flow cytometry. Twelve (41.4%) of the 29 patients developed moderate or severe disorders in liver function within 6 months after LDLT. Eight of the 12 patients (overall incidence of 27.6%) suffering from liver function disorder were diagnosed on the basis of liver biopsy results as having mild or moderate acute rejection. However, only 4 of the 12 patients (overall incidence of 13.8%) showed remarkable proliferation of CD8+ T cells in association with CD25 expression on antidonor CFSE-MLR. The other eight patients were eventually diagnosed as having recurrence of original hepatitis, drug-induced hepatotoxicity, or congestion of the anterior segment of the liver allograft by further extensive examinations or in retrospect. The results of CFSE-MLR assays, which could be used for rigorously monitoring rejection, provided evidence of low incidence of acute rejection after LDLT.

Retinoic Acid Inhibits the Proliferative Response Induced by CD40 Activation and Interleukin-4 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavorable prognosis. Here, we show that retinoic acid (RA) isomers significantly inhibit the proliferation of both primary MCL cultures (n = 7) and established cell lines (Granta 519 and SP-53) as shown by [(3)H]thymidine uptake and carboxyfluorescein diacetate succinimidyl ester labeling coupled with cyclin D1 staining. RA induces cell accumulation in G(0)-G(1) together with a marked up-regulation of p27(Kip1) by inhibiting ubiquitination and proteasome-dependent degradation of the protein. The p21(Cip1) inhibitor was also up-regulated by RA in Granta 519 cells, whereas the expression of cyclin D1 is unaffected. Most of RA-induced p27(Kip1) was bound to cyclin D1/cyclin-dependent kinase 4 complexes, probably contributing to the decreased cyclin-dependent kinase 4 kinase activity and pRb hypophosphorylation observed in RA-treated cells. Experiments with receptor-selective ligands indicate that RA receptor alpha cooperates with retinoid X receptors in mediating RA-dependent MCL cell growth inhibition. Notably, RA isomers, and particularly 9-cis-RA, also inhibited the growth-promoting effect induced in primary MCL cells by CD40 activation alone or in combination with interleukin-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results therefore further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds, our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting.