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Cytometric Analysis (cytometric + analysis)
Kinds of Cytometric Analysis Selected AbstractsScreening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escapeCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2005E. Ortona Summary The worldwide problem of chronic Echinococcus granulosus disease calls for new parasite-derived immunomodulatory molecules. By screening an E. granulosus cDNA library with IgG4 from patients with active cystic echinococcosis, we identified a cDNA that encodes a predicted partial protein that immunofluorescence studies localized in the protoscolex tegument and on the germinal layer of cyst wall. We named this protein EgTeg because the 105 amino acid sequence scored highest against a family of Schistosoma tegumental proteins. Evaluating the role of EgTeg in the human early inflammatory response we found that EgTeg significantly inhibited polymorphonuclear cell (PMN) chemotaxis. Cytometric analysis of intracellular cytokines disclosed a significantly higher percentage of cells producing IL-4 than IFN-, (P = 0·001, Student's t-test) in T lymphocytes from patients with cystic echinococcosis stimulated with EgTeg. EgTeg induced weak Th1-dependent proliferation in 42% of patients' peripheral blood mononuclear cells. In immunoblotting (IB) analysis of total IgG and IgG subclass responses to EgTeg in patients with cystic echinococcosis, patients with other parasitoses, patients with cystic lesions and healthy controls, total IgG specific to EgTeg yielded high sensitivity (73%) but low specificity (44%) precluding its use in immunodiagnosis. Conversely, IgG4 specific to EgTeg gave acceptable sensitivity (65%) and high specificity (89%) suggesting its use in immunodiagnosis to confirm ultrasound documented cysts suggestive of E. granulosus. Because the new tegumental antigen EgTeg inhibits chemotaxis, induces IL-4-positive T lymphocytes and noncomplement fixing antibodies (IgG4) it is an immunomodulatory molecule associated with chronic infection. [source] Phenotypic classification of human CD8+ T,cells reflecting their function: inverse correlation between quantitative expression of CD27 and cytotoxic effector functionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2004Hiroko Tomiyama Abstract Phenotypic classification of human CD8+ T,cells using three cell surface markers, CD27, CD28 and CD45RA, was recently suggested to be useful for identification of naive, memory and effector CD8+ T,cells. However, it still remains unclear whether such classification precisely reflects functional classification of CD8+ T,cells. To clarify this, we characterized each CD27CD28CD45RA subset of total and human cytomegalovirus (HCMV)-specific CD8+ T,cells by analyzing the expression of perforin and two chemokine receptors, CCR5 and CCR7, as well as their function. An inverse correlation between perforin and CD27 expression was found in all four CD28CD45RA subsets. Therefore, to achieve a phenotypic classification of CD8+ T,cells that moreprecisely reflects their function, the CD27+ subset was divided into CD27low and CD27high subsets based on the expression level of CD27. Functional and flow cytometric analyses of CD27CD28CD45RA subsets showed that this phenotypic classification reflects functional classification of CD8+ T,cells. HCMV-specific CD8+ T,cells from healthy HCMV-seropositive individuals were predominantly found in effector and memory/effector subsets, indicating that HCMV-specific effector CD8+ T,cells are actively induced by HCMV replication in healthy HCMV carriers. Phenotypic analyses of CD8+ T,cells using this classification will enable the characterization of antigen-specific CD8+ T,cells. [source] Methyl tert -butyl ether (MTBE)-induced cytotoxicity and oxidative stress in isolated rat spermatogenic cellsJOURNAL OF APPLIED TOXICOLOGY, Issue 1 2007Dongmei Li Abstract Methyl tert -butyl ether (MTBE) is a class of synthetic organic chemical. In the USA, MTBE pollution is regarded as a serious environmental problem. The objective of the present study was to investigate the cytotoxic effects and oxidative stress induced by MTBE in isolated rat spermatogenic cells. In cytotoxic experiments, spermatogenic cells isolated from the testes of adult Sprague-Dawley rats by a mechanical procedure without the use of trypsin were incubated with medium alone (control), 0.5, 5, 50 mm MTBE, respectively, for 6, 12 and 18 h. MTT assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI) and flow cytometric analyses were used. In oxidative stress experiments, the spermatogenic cells were incubated with medium alone (control) and with 0.5, 50 ,m, 5 mm MTBE. For 1, 2, 6, 12, 18 h incubation, ROS production was tested using a 2,,7,-dichlorofluorescein diacetate (DCHF-DA) probe; for 1, 3, 6, 12, 18 h incubation, cytosolic superoxide dismutase (SOD) and extracellular SOD (SODEX) activity was assessed; and for 18 h incubation, lipid peroxidation was assessed. The results showed that MTBE at high doses significantly decreased the spermatogenic cell viability and increased plasma membrane damage and the ratio of necrotic cells compared with the control. Assessment of the MTBE-induced oxidative stress revealed that MTBE increased the production of reactive oxygen species (ROS) and enhanced lipid peroxidation. In addition, although SODEX activity increased at a high dose level, cytosolic SOD activity decreased. These results suggest that an increase of MTBE-induced ROS production and an enhancement of membrane lipid peroxidation may play an important role in its cytotoxicity in isolated rat spermatogenic cells. Copyright © 2006 John Wiley & Sons, Ltd. [source] PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005Nabanita S Datta PhD Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [source] BT-IgSF, a novel immunoglobulin superfamily protein, functions as a cell adhesion moleculeJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Hideki Harada BT-IgSF is a newly identified cell surface glycoprotein belonging to the immunoglobulin superfamily (IgSF). We have previously shown that the expression of the BT-IgSF gene was highly restricted to brain and testis, and its transcript was detected in both neurons and glial cells. In this study, to explore its function, we generated cells overexpressing BT-IgSF proteins and analyzed their phenotypes. We found that the constitutive expression of BT-IgSF in the myeloid leukemia cell line TF-1 -fms did not alter the growth rates, but caused the formation of large cell aggregates. The cell aggregates were also observed with mutant BT-IgSF lacking its cytoplasmic tail, the amino acid sequences of which were highly conserved among the BT-IgSF subgroup proteins. The neutralizing antibody to ,1 integrin did not diminish the cell aggregate formation. These results indicate that BT-IgSF functions as a cell adhesion molecule, that its cytoplasmic tail is not essential for the function, and that ,1 integrin is not involved in the function. We confirmed the cell adhesion function using NIH/3T3 fibroblastic cells expressing BT-IgSF in an inducible system. Flow cytometric analyses with the cells demonstrated that the cell aggregation mediated by BT-IgSF was through homophilic molecular interaction, and in a Ca2+/Mg2+ -independent manner. Coupled with its restricted pattern of the expression, the cell adhesion-inducing function of BT-IgSF suggests a role of the cell surface molecule in the development/function of the central nervous system and spermatogenesis. © 2005 Wiley-Liss, Inc. [source] Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycleJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2003Gavin Brooks ABSTRACT Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders. [source] Expression of receptor activator of nuclear factor-,B ligand by B cells in response to oral bacteriaMOLECULAR ORAL MICROBIOLOGY, Issue 3 2009X. Han Introduction:, We investigated receptor activator of nuclear factor-,B ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Methods:, Expression of messenger RNA transcripts (tumor necrosis factor-,, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription,polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. Results:, The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans -immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans -immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. Conclusion:, This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease. [source] Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosomeANDROLOGIA, Issue 1 2010N. Falzone Summary Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false-positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) were compared for their ability to stain nonviable cells post-fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7-AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7-AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore-induced acrosome reaction. [source] Acquired but reversible loss of erythrocyte complement receptor 1 (CR1, CD35) and its longitudinal alteration in patients with severe acute respiratory syndromeCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2005F. S. Wang Summary This longitudinal study investigates the change of erythrocyte complement receptor (E-CR1) expression in patients with severe acute respiratory syndrome (SARS). Circulating E-CR1 expression was semiquantified by flow cytometric analyses in 54 SARS patients and in 212 healthy individuals as a control. Since E-CR1 expression is influenced by the genetic polymorphisms in the CR1 gene, a major genetic polymorphism located within intron 27 of the CR1 gene was simultaneously analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed that the expression level of E-CR1 (referred to as net fluorescence intensity values, NFI) was statistically correlated with the relevant genetic genotypes among the Chinese population including the healthy individuals (NFI: 5·14 ± 0·82, 3·57 ± 0·66 and 2·67 ± 0·32 for HH, HL and LL genotypes, respectively) and SARS patients (NFI: 3·52 ± 0·91 and 2·63 ± 0·70 for HH and HL genotypes, respectively). Interestingly, the expression density of E-CR1 was found to fall significantly during the initiation and progressive phases (weeks 1 and 2 after the disease onset) and gradually returned close to normal through their whole convalescent phase (beginning from weeks 2 or 3 to weeks 7 or 8) in SARS patients irrespective CR1 genotype. In conclusion, our findings, at least, suggest that E-CR1 is likely involved in immune pathogenesis of SARS disease. [source] Perinatal development of the rat kidney: Apoptosis and epidermal growth factorCONGENITAL ANOMALIES, Issue 3 2003Toshiya Okada ABSTRACT, Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase,mediated d,UTP,biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney. [source] Clinical utility of CD23 and FMC7 antigen coexistent expression in B-cell lymphoproliferative disorder subclassificationCYTOMETRY, Issue 1 2002Ejaz Ahmad Abstract Background: CD23 and FMC7 are normal B-cell antigens utilized during diagnostic immunophenotyping of suspected lymphoproliferative disorders. However, the diagnostic utility of coexistent antigenic expression patterns with simultaneous two-color staining and flow cytometric analysis has not been studied extensively. Methods: Using multiparameter flow cytometry, we evaluated the expression pattern of FMC7 and CD23 in 218 cases of B-cell lymphoma from blood and bone marrow specimens. Results: The CD23(+)/FMC7(-) pattern was the most common pattern in patients with chronic lymphocytic leukemia and related variants. The widest variation of patterns was found in patients with follicular cell lymphoma, large cell lymphoma, and Waldenström's macroglobulinemia, a lymphoplasmacytoid disorder, although most cases expressed the CD23-/FMC7(+) pattern. The CD23 and FMC7 antigen, along with the CD5 coexpression pattern, provides critical adjunctive data. These data allow accurate classification of the majority of cases, thereby providing a key aspect of a reliable diagnostic algorithm. The CD23 and FMC7 antigen expression pattern, along with selected other antigens, was predictive of subtypes in >95% of lymphoproliferative cases and narrowed the differential diagnosis in the remaining cases. Conclusion: The flow cytometric CD23/FMC7 expression pattern achieved by multicolor immunophenotyping facilitates accurate and reproducible classification of B-cell lymphomas and has diagnostic utility. Cytometry (Clin. Cytometry) 50:1,7, 2002. © 2002 Wiley-Liss, Inc. [source] Novel application of flow cytometry: Determination of muscle fiber types and protein levels in whole murine skeletal muscles and heartCYTOSKELETON, Issue 12 2007Connie Jackaman Abstract Conventional methods for measuring proteins within muscle samples such as immunohistochemistry and western blot analysis can be time consuming, labor intensive and subject to sampling errors. We have developed flow cytometry techniques to detect proteins in whole murine heart and skeletal muscle. Flow cytometry and immunohistochemistry were performed on quadriceps and soleus muscles from male C57BL/6J, BALB/c, CBA and mdx mice. Proteins including actins, myosins, tropomyosin and ,-actinin were detected via single staining flow cytometric analysis. This correlated with immunohistochemistry using the same antibodies. Muscle fiber types could be determined by dual labeled flow cytometry for skeletal muscle actin and different myosins. This showed similar results to immunohistochemistry for I, IIA and IIB myosins. Flow cytometry of heart samples from C57BL/6J and BALB/c mice dual labeled with cardiac and skeletal muscle actin antibodies demonstrated the known increase in skeletal actin protein in BALB/c hearts. The membrane-associated proteins ,-sarcoglycan and dystrophin could be detected in C57BL/6J mice, but were decreased or absent in mdx mice. With the ability to label whole muscle samples simultaneously with multiple antibodies, flow cytometry may have advantages over conventional methods for certain applications, including assessing the efficacy of potential therapies for muscle diseases. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Assessment of myosin II, Va, VI and VIIa loss of function on endocytosis and endocytic vesicle motility in bone marrow-derived dendritic cellsCYTOSKELETON, Issue 10 2007Jeffrey P. Holt Abstract An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on ratesof endocytic vesicle movement. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Acute exposure of human lung cells to 1,3-butadiene diepoxide results in G1 and G2 cell cycle arrestENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2005Michael Schmiederer Abstract 1,3-butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2 -treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2 -treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 ,M BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2 -treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2 -treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2 -treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21cip1 was observed in BDO2 -treated cells, suggesting that the cell cycle arrest was p21cip1 -mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Effects of nonylphenol, bisphenol a, and their mixture on the viviparous swordtail fish (Xiphophorus helleri)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2001Hyeong-Il Kwak Abstract A number of fish species have been used for studies on endocrine disrupting chemicals (EDCs). However, despite the widespread use of oviparous fish, relatively little attention has been given to viviparous species. This study investigated the effects of EDCs in a viviparous fish and examined the possible usefulness of the fish as an alternative model for the studies on EDCs. Swordtails (Xiphophorus helleri) were exposed to nonylphenol (NP), bisphenol A (BPA), and their mixture. Both short-term (3-d) and relatively long-term (60-d) exposures were carried out using adult male and 30-d-old juvenile fish, respectively. Following the short-term exposure, both NP and BPA caused vitellogenin mRNA expression. Flow cytometric analysis and terminal deox-ynucleotidyl transferase assay on the testes of treated fish indicated reproductive damage. Histopathological analysis found degenerative and necrotic cells in seminiferous tubules following the exposure to 100 ppb NP. The testes with lesions were also associated with highly suppressed spermatogenesis. Following the long-term exposure, both NP and BPA exposures significantly affected the growth of swordtails. In all cases, the results showed that the mixture was always more potent than a single chemical and that swordtail fish can be a useful model for the study of endocrine disruptors. [source] An immunodeficiency in Fell ponies: a preliminary study into cellular responsesEQUINE VETERINARY JOURNAL, Issue 7 2001S. C. BELL Summary A putative immunodeficiency, causing mortality in UK Fell pony foals (Fell pony syndrome), was studied in affected foals and compared with healthy, age-matched foals. Differential cell counts of peripheral blood indicated that the syndrome foals were lymphopenic (P<0.05). Flow cytometric analysis of circulating leucocytes showed a reduced MHC II expression (P<0.01) on lymphocytes but not on polymorphonuclear cells in affected foals. There were no changes in the percentages of CD4+ or CD8+ T cells. There was an increased (P<0.05) expression of CD11a/18 by the lymphocytes of the syndrome foals, compared to the control foals, which is probably a response to systemic bacterial infections. The syndrome foals' lymphocytes responded to mitogens (PHA, ConA, PWM) at normal levels. The data do not conform to any known immunodeficiencies identified in any other species. Further analyses will be required, particularly on bone marrow function. [source] Neonatal isoimmune thrombocytopenia caused by type I CD36 deficiency having novel splicing isoforms of the CD36 geneEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2008Takeshi Taketani Abstract Neonatal alloimmune thrombocytopenia (NAIT) occurs because of transplacentally acquired maternal platelet alloantibodies. Most of the alloantibodies are against human platelet antigens, but the alloantibody against CD36 is rare. A full-term female baby was delivered by a mother who experienced two spontaneous abortions. The baby had thrombocytopenia with cephalhematoma. The platelet count increased by immunoglobulin therapy (400 mg/kg) for 3 d. Platelet antibody was detected in the postpartum maternal serum. The specificity of the antibody directed against platelets was identified as anti-Naka (CD36). Flow cytometric analysis showed no expression of CD36 in both platelets and monocytes from mother. Mutation analysis revealed two different splicing isoforms of maternal CD36 mRNA. One allele was exon 4 skipping, another was exon 9 skipping, both of which led to a frameshift and produced a truncated CD36 protein. These results indicate that NAIT is caused by maternal CD36 deficiency having CD36 splicing abnormalities. [source] Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patientsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2007Andrea De Maria M. D. Abstract Hepatitis C virus (HCV) readily establishes high-level lifelong persistent infection in the majority of immunocompetent adults with failure of HCV-specific CD8+ CTL to clear viral replication. Virus-induced conditioning of innate immune responses is a possible mechanism that may contribute to the impairment of virus-specific CD8+ CTL responses. Here, we analyzed whether triggering of NK cell receptor expression and function is affected during chronic viremic HCV infection. Flow cytometric analysis of purified resting peripheral NK cells showed no evidence of NK cell activation, while analysis of natural cytotoxicity receptors (NCR) showed that NK cells from HCV-infected patients had selective increased expression of NKp30 and NKp46. NK cells had corresponding conserved cytotoxic activity against all targets with the exception of HepG2 hepatoma cells. Freshly separated NK cells from HCV patients showed significant production of IL-10 and normal concentrations of IFN-, upon cell-mediated triggering. Thus, increased expression of NKp30 during HCV infection with increased IL-10 production could contribute, once NK cells localize in the liver, to a NK-DC crosstalk leading to skewing of subsequent adaptive immune responses and lack of virus control. [source] Treatment of neonatal mice with Flt3 ligand leads to changes in dendritic cell subpopulations associated with enhanced IL-12 and IFN-, productionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2004Sabine Vollstedt Abstract Treatment with the hematopoietic growth factor Flt3 ligand (FL) increases DC numbers in neonatal mice and enhances their resistance against intracellular pathogens. Flow cytometric analysis showed the presence of conventional DC (cDC) and plasmacytoid pre-DC (pDC) in neonatal spleens from untreated and FL-treated mice. CD8, and MHC class,II expression on cDC and pDC was higher on DC from FL-treated mice than on DC from control littermates. After FL treatment, two additional subpopulations of DC-lineage cells were found that were able to produce IL-12 and IFN-,. The IL-12 production of cDC from FL-treated animals was more than 50-fold increased and their ability to stimulate T,cell proliferation was also increased. We conclude that the enhanced resistance against intracellular pathogens was due to increased numbers of DC-lineage cells and their increased ability to produce the essential cytokines. [source] Neurosphere generation from dental pulp of adult rat incisorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2008Ryo Sasaki Abstract Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nervous system disorders. Here we report that adult rat dental pulp cells have the ability to form neurospheres when cultured in serum-free culture medium on super-hydrophilic plates. The cells within small spheres continued to grow, and the dental pulp-derived cells generated large spheres. Sphere formation was dependent on exogenously supplied basic-fibroblast growth factor, but not on epidermal growth factor, and the formation and growth of dental pulp-derived spheres were negatively regulated by transforming growth factor-,. Plating cells that were dissociated from spheres on an adhesive substrate resulted in differentiation into Tuj1- and MAP2-positive neuronal cells. Analysis of the three-dimensional structure of dental pulp-derived spheres shows that they contained nestin-positive progenitors, Tuj1-positive neuronal cells and S100-positive glial cells. We found that spheres contained CD81 (TAPA1) and nestin double-positive cells, and identified a small population of CD81 and nestin double-positive cells in the odontoblast layer of the dental pulp. Flow cytometric analysis showed that CD81-positive cells were enriched in the spheres compared with the dental pulp tissue. Bromodeoxyuridine (BrdU) staining showed that nestin- and BrdU-positive cells were located only in the apical portion of the dental pulp, and the apical portion produced a large number of large-sized spheres. These data suggest that the CD81 and nestin double-positive cells localized in the odontoblast layer of the apical portion of the dental pulp may have the ability to grow and form neurospheres. [source] Terrein inhibits keratinocyte proliferation via ERK inactivation and G2/Mcell cycle arrestEXPERIMENTAL DERMATOLOGY, Issue 4 2008Dong-Seok Kim Abstract:, Terrein, a fungal metabolite, has been recently shown to have a strong antiproliferative effect on skin equivalents. In the present study, we further investigated the effects of terrein on the possible signalling pathways involved in the growth inhibition of human epidermal keratinocytes by examining the regulations of extracellular signal-regulated protein kinase (ERK) and of the Akt pathway by terrein. It was observed that ERK was inactivated by terrein and that keratinocyte proliferation was inhibited, whereas Akt was unaffected. The inhibition of the ERK pathway by U0126 (a specific ERK inhibitor) also had a dose-dependent antiproliferative effect on human keratinocytes. These results indicate that ERK inhibition is involved in keratinocyte growth inhibition by terrein. Moreover, flow cytometric analysis showed that terrein inhibits DNA synthesis, as evidenced by a reduction in the S phase and an increase in the G2/M phase of the cell cycle. Thus, we next examined changes in the expressions of G2/M cell cycle-related proteins. Terrein was found to downregulate cyclin B1 and Cdc2 without Cdc2 phosphorylation, but upregulated p27KIP1 (p27), a known inhibitor of cyclin-dependent kinase. These results suggest that terrein reduces human keratinocyte proliferation by inhibiting ERK and by decreasing the expressions of cyclin B1 and Cdc2 complex. [source] In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cellsEXPERIMENTAL DERMATOLOGY, Issue 6 2003Bo Bang Abstract:,In vitro studies with human cell lines have demonstrated that the death receptor Fas plays a role in ultraviolet (UV)-induced apoptosis. The purpose of the present study was to investigate the relation between Fas expression and apoptosis as well as clustering of Fas in human epidermis after a single dose of UVB irradiation. Normal healthy individuals were irradiated with three minimal erythema doses (MED) of UVB on forearm or buttock skin. Suction blisters from unirradiated and irradiated skin were raised, and Fas, FasL, and apoptosis of epidermal cells were quantified by flow cytometry. Clustering of Fas was demonstrated by confocal laser scanning microscopy on cryostat sections from skin biopsies. Soluble FasL in suction blister fluid was quantified by ELISA. Flow cytometric analysis demonstrated increased expression intensity of Fas after irradiation, with 1.6-, 2.2- and 2.7-fold increased median expression at 24, 48 and 72 h after irradiation, respectively (n = 4). Apoptosis was demonstrated by the TUNEL reaction, and the maximum of apoptotic cells was detected at 48 h after irradiation. Double-staining for Fas and TUNEL showed that apoptosis was restricted to the Fas-positive epidermal subpopulation, but there was no correlation between the intensities of Fas expression and TUNEL reaction. Median expression intensity of FasL-positive cells transiently decreased to 0.9- and 0.8-fold of the preirradiation respective level after 24 h and 48 h, respectively, and returned to the respective preirradiation level at 72 h after irradiation (n = 4). Concentrations of soluble FasL in suction blister fluid from UVB-irradiated skin did not differ from those in unirradiated skin (n = 5). Confocal laser scanning microscopy showed a rapid clustering of Fas within 30 min after irradiation. A simultaneous clustering of the adapter signalling protein FADD suggested that Fas clustering has a functional significance. Our results are in accordance with previous findings from in vitro studies, and suggest that Fas is activated in vivo in human epidermis after UVB exposure. [source] Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipinFEBS JOURNAL, Issue 21 2009Patrice X. Petit BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices ,H6 and ,H7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c -releasing activity. In the present study, we focused on the interaction between the ,H6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the ,H6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of ,H6 into the phospholipid monolayer. The modification of two charged amino acid residues in ,H6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and ,H6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that ,H6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via ,H6. [source] Membrane orientation of laminin binding proteinFEBS JOURNAL, Issue 18 2003An extracellular matrix bridging molecule of Leishmania donovani Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source] Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect on Candida albicansFEBS JOURNAL, Issue 16 2001Dae-Hee Kim Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization. [source] The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococciFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2008Jan Kolberg Abstract Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions. [source] Flow cytometric analysis of the localization of Helicobacter pylori antigens during different growth phasesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2001Kristina Blom Abstract Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N -acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori. [source] Near-diploid and near-triploid human sporadic colorectal adenocarcinomas differ for KRAS2 and TP53 mutational statusGENES, CHROMOSOMES AND CANCER, Issue 2 2003Walter Giaretti Mutations of the KRAS2 protoncogene and inactivation of the TP53 oncosuppressor gene have been suggested to contribute to chromosomal instability (CIN) and aneuploidy in colorectal cancer (CRC). Previous work has also shown that the degree of DNA ploidy [DNA index (DI)], as obtained by flow cytometry in CRC, is non-randomly distributed and, in particular, that DI near-diploid and near-triploid values are well separated by a low-probability valley region. At present, it is not known whether a relationship exists between DI and the mutational status of KRAS2 and TP53. Multiple samples obtained from 35 human sporadic CRCs have been used to provide nuclei suspensions for flow cytometric analysis and sorting of specific DI subpopulations. Sorted nuclei were then used to analyze the high-microsatellite-instability (MSI-H) phenotype and the mutation spectrum of the KRAS2 and TP53 genes. A single MSI-H case was detected. There were 6 DNA diploid (DI = 1) and 29 aneuploid (DI , 1) CRCs, with the DI aneuploid cases non-randomly subdivided in 9 near-diploid (DI , 1 and DI , 1.4), 8 near-triploid (1.4 < DI < 1.6), and 12 high-aneuploid (DI , 1.6) cases. Proximal CRCs were more often DNA diploid and near-diploid than distal ones, and Dukes' C cases were more commonly high-aneuploid than Dukes' B. Moreover, the incidence of mutations of the KRAS2 and TP53 genes was lowest among the DNA near-triploid subpopulations and highest among the near-diploid ones. We suggest that DNA near-diploid and near-triploid subpopulations in human sporadic CRC reflect different genetic mechanisms of CIN and have a potentially different clinical behavior. © 2003 Wiley-Liss, Inc. [source] Can flow cytometrically determined DNA ploidy and S-phase fraction predict regional metastasis in squamous cell carcinoma of the oral cavity?HEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 2 2002Ryoichi Oya DDS Abstract Background The value of flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) as an indicator of regional metastasis in oral cancer is currently being debated. Intratumoral heterogeneity makes this problem complex. Methods Intratumoral DNA ploidy heterogeneity and intratumoral SPF variation were examined using multiple specimens from 31 surgically resected specimens taken from patients with oral cancer without preoperative therapy. Flow cytometric analysis of single biopsy specimens from 79 patients with oral cancer was also undertaken to ascertain their value as indicators of regional metastasis. Results Forty-five percent (14 of 31) of tumors showed intratumoral ploidy heterogeneity. Intratumoral SPF variation in the 31 tumors ranged from 0.2% to 6.9% (mean, 3.3%). Multivariate analysis showed that a SPF greater than 27% was the most important parameter for predicting regional metastasis. Conclusions DNA ploidy is heterogeneous within a tumor, whereas SPF is relatively stable and can be correlated with regional metastasis in oral cancer. © 2002 John Wiley & Sons, Inc. [source] Patients with bisphosphonates-associated osteonecrosis of the jaw have reduced circulating endothelial cellsHEMATOLOGICAL ONCOLOGY, Issue 4 2007A Allegra Abstract Osteonecrosis of the jaws (ONJ) associated with the use of bisphosphonates is a newly described entity. To elucidate the mechanism leading to ONJ and to test the hypothesis that in patients with ONJ the bisphosphonates may interfere with endothelial cell proliferation, using flow cytometric analysis we evaluated the number of circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) in eight patients with bisphosphonate treatment and osteonecrosis, eight multiple myeloma (MM) patients with bisphosphonates treatment without ONJ and five normal subjects. MM patients showed an increase of CD34+ cells with respect the control subjects and ONJ subjects. EPCs and CECs were higher in MM patients compared to controls and ONJ patients. ONJ patients showed a decrease of EPCs compared to control subjects while CECs were similar to the controls group. Our results seem to show the possibility that bisphosphonates could have a antiangiogenic effect and a suppressive effect on CECs of patients with ONJ. Copyright © 2007 John Wiley & Sons, Ltd. 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