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Intracellular Expression (intracellular + expression)
Selected AbstractsCTLA-4 expression in T cells of patients with atopic dermatitisPEDIATRIC ALLERGY AND IMMUNOLOGY, Issue 5 2005Sung Yon Choi Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is a surface molecule of activated T cells with sequence homologous to CD28, and may act as a negative regulator of T-cell activation. In murine animal models, cross-linkage of CTLA-4 molecules on the cell surface results in decreased T-cell proliferation, accompanied by increased interleukin (IL)-2 production and apotosis. To clarify the activation of peripheral blood T cells, we studied the CTLA-4 expression in 32 patients with atopic dermatitis who visited our institution, and 19 normal children who visited for pre-operative laboratory examination were used as normal controls. Whole blood was obtained from all subjects and stained with anti-CD3, anti-CD4, anti-CD8 monoclonal antibodies (mAb). After erythrocyte lysis with lysing solution, the cells were stained with anti-CTLA-4 mAb, and stained cells were analysed by fluorescence-activated cell sorter (FACScan) flow cytometer. Intracellular expression of CTLA-4 was significantly upregulated in peripheral blood CD3+ T cells (36.8%), CD4+ T cells (21.7%) and CD8+ T cells (18.7%) of patients with atopic dermatitis, compared with normal control (18.3%, 9.7%, 9.8%; respectively). Furthermore, CTLA-4-positive CD3+ T cells in patients with severe atopic dermatitis were significantly higher compared with milder group (42.8% vs. 32.2%). However, no significant difference was obtained in CD4+ and CD8+ T cells. Mean percentage of T cells expressing CTLA-4 in patients with atopic dermatitis was higher than the control group. These observations suggest the possibility that the disease activity can be correlated with the CTLA-4 level. [source] Monocytes and T lymphocytes contribute to a predominance of interleukin 6 and interleukin 10 in systemic lupus erythematosus,CYTOMETRY, Issue 4 2009Susana Mellor-Pita Abstract Objective To investigate the contribution of T lymphocytes and monocytes to cytokine production in systemic lupus erythematosus (SLE). Methods Forty-five SLE patients and 19 healthy volunteers were included. Serum levels of tumor necrosis factor alpha (TNF,), interferon gamma (IFN,), interleukin (IL)-6, and IL10 were quantified by ELISA. The cytokine production capacities of peripheral blood mononuclear cells were assessed by culturing in vitro with PMA+Ionomycin or LPS. The intracellular cytokine expression was measured by flow cytometry in T lymphocytes and monocytes, respectively. The influence of the disease activity (measured as the SLE-disease activity index; SLEDAI) and the treatment the patients were receiving was evaluated. Results Serum IL10, IL6, and TNF, levels were increased in patients (P , 0.01), and a higher spontaneous (without stimuli) intracellular expression of IL10 in CD4+ and CD8+ T lymphocytes (P < 0.05) and of IL6 in monocytes (P = 0.01) were found. After stimulation, patients presented a higher percentage of CD4+ and CD8+ T lymphocytes producing IL4 and IL10 (P , 0.01), and of monocytes producing IL6 (P = 0.04) and IL10 (P = 0.008). The SLEDAI score was positively correlated with the percentage of CD4+IL10+ and CD8+IL10+ T lymphocytes (P < 0.01), and inversely correlated with CD8+TNF,+ (P= 0.02), CD4+IFN,+ (P = 0.04) and CD8+ IFN,+ (P = 0.002) T lymphocytes. Patients receiving high dose prednisone produced higher IL10, but they also were the patients with a more active disease. Conclusion Monocytes and T lymphocytes (CD4+ and CD8+) contribute to an overproduction of IL6 and IL10 in SLE; this correlates with the disease activity but is independent of the treatment the patients are receiving. © 2009 Clinical Cytometry Society [source] Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumorsCYTOMETRY, Issue 1 2006Stanley Shackney Abstract Background: Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. Methods and results: We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Conclusions: Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. © 2005 Wiley-Liss, Inc. [source] Changes in the cytologic distribution of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) during in vivo and in vitro mouse mammary epithelial cell expression and differentiationDEVELOPMENTAL DYNAMICS, Issue 1 2002Catherine B. Kirn-Safran Abstract HIP/RPL29 is a small, highly basic, heparin/heparan sulfate interacting protein identical to ribosomal protein L29 and present in most adult epithelia. In the present study, we show that mouse HIP/RPL29 is ubiquitously present in adult mammary epithelia and is significantly increased during pregnancy and lactation. We observed for the first time that HIP/RPL29 intracellular expression and distribution varies, depending on the growth/differentiation state of the luminal epithelium. HIP/RPL29 was detected at low levels in mammary glands of virgin animals, increased markedly during lactation, and was lost again during involution. HIP/RPL29, preferentially found in the expanded cytoplasm of mature epithelial cells secreting milk, is present also in the nucleus of proliferating and differentiating ductal and alveolar elements. We used COMMA-D cells as an in vitro model for mammary-specific differentiation and examined similar intracellular redistribution of HIP/RPL29 associated with functional differentiation. However, no changes in HIP/RPL29 expression levels were detected in response to lactogenic hormones. Finally, the cellular distribution of HIP/RPL29 in both nuclear and cytoplasmic compartments was confirmed by transfecting a normal mammary epithelial cell line, NMuMG, with a fusion protein of HIP/RPL29 and EGFP. Collectively, these data support the idea that HIP/RPL29 plays more than one role during adult mammary gland development. © 2001 Wiley-Liss, Inc. [source] Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial,neuronal culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008Takayuki Nakagawa Abstract Glutamate uptake by the Na+ -dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial,neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1,EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N -methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC50 value of glutamate was 240 ,m. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na+ -free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L- trans -pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na+/K+ -ATPase inhibitor, ouabain, and the Na+ ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca2+signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na+ influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes. [source] Diverting a protein from its cellular location by intracellular antibodiesFEBS JOURNAL, Issue 4 2000The case of p21Ras We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic. [source] Activated monocytes and platelet-monocyte aggregates in patients with sickle cell disease*INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 2 2002TED WUN Tumour necrosis factor-, (TNF-,) and interleukin-1, (IL-1,) increase endothelial surface receptors that mediate the adherence of sickle erythrocytes to the endothelium. Increased circulating levels of these cytokines have been found in patients with sickle cell disease (SCD). Monocytes are a source of both of these inflammatory mediators; we therefore determined whether circulating monocytes were activated in SCD, as defined by intracellular expression of these cytokines. Blood was also assayed for the presence of platelet,monocyte aggregates (PMAs), as platelet adherence is one possible mechanism for monocyte activation. The median percentages of monocytes expressing intracellular TNF-, and IL-1, in SCD patients were 6.8 (2.8,17.3) [median (range)] and 14.1 (1.3,44.8), respectively. In African-American controls the corresponding percentages were 0.3 (0.1,0.5) and 0.4 (0.1,3.0), and in Caucasians 0.2 (0.1,0.5) and 0.8 (0.8,1.9) (P < 0.001, Kruskal,Wallis). The mean percentage (± SD) of PMA was 14.0 ± 8.3 for Caucasian controls, 25.7 ± 7.3 for African-American controls, and 45.7 ± 21.6 for patients with SCD (P < 0.001, RM ANOVA; P < 0.05, Newman,Keuls posthoc test). We conclude that there are increased circulating PMAs and monocyte activation in patients with SCD. [source] Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2000Homayoun H. Zadeh Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells. However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-,, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24,48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7%±0.3), IFN-,(1.8%±0.5), IL-4 (1.0%±0.2) and IL-10 (1.5%±0.5). These data indicated that only 2,5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcomitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69+) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA+ionomycin stimulation. Results showed that number of IL-2+ and IFN-,+ T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in response to PMA+ionomycin. Conversely, the proportions of T cells expressing IL-4 or IL-10 were between 35% and 90% of those following stimulation with PMA+ionomycin. Hence, A. actinomycetemcomitans appears to more preferentially induce T cells [source] Gene therapy for HIV/AIDS: the potential for a new therapeutic regimenTHE JOURNAL OF GENE MEDICINE, Issue 8 2003Greg Fanning Abstract Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). HIV/AIDS is a disease that, compared with the not so distant past, is now better held in check by current antiretroviral drugs. However, it remains a disease not solved. Highly active antiretroviral therapy (HAART) generally uses two non-nucleoside and one nucleoside reverse transcriptase (RT) inhibitor or two non-nucleoside RT and one protease inhibitor. HAART is far more effective than the mono- or duo-therapy of the past, which used compounds like the nucleoside reverse transcriptase inhibitor AZT or two nucleoside reverse transcriptase inhibitors. However, even with the relatively potent drug cocktails that comprise HAART, there are the issues of (i) HIV escape mutants, (ii) an apparent need to take the drugs in an ongoing manner, and (iii) the drugs' side effects that are often severe. This review speaks to the potential addition to these potent regimens of another regimen, namely the genetic modification of target hematopoietic cells. Such a new treatment paradigm is conceptually attractive as it may yield the constant intracellular expression of an anti-HIV gene that acts to inhibit HIV replication and pathogenicity. A body of preclinical work exists showing the inhibition of HIV replication and decreased HIV pathogenicity by anti-HIV genetic agents. This preclinical work used hematopoietic cell lines and primary cells as the target tissue. More recently, several clinical trials have sought to test this concept in vivo. Copyright © 2003 John Wiley & Sons, Ltd. [source] Recipient CTLA-4 +49 G/G Genotype Is Associated with Reduced Incidence of Acute Rejection After Liver TransplantationAMERICAN JOURNAL OF TRANSPLANTATION, Issue 12 2003Philip de Reuver The aim of this pilot study was to investigate whether acute rejection after liver transplantation is associated with known single-nucleotide polymorphisms (SNPs) in the CD86- and CTLA-4 genes of liver-transplant donors and recipients. Single nucleotide polymorphisms were determined in 135 liver transplant recipients and in 73 donors. Acute rejection was not associated with CD86 + 1057 G/A genotype distributions in donors and in recipients. In univariate analysis recipient CTLA-4 ,318 G/T and + 49 A/G genotype distributions were both weakly associated with acute rejection. Multivariate analysis revealed that the CTLA-4 + 49 SNP, but not the ,318 SNP, was independently of other risk factors associated with acute rejection. Only one out of 13 CTLA-4 + 49 G-homozygotes (8%) experienced acute rejection(s) compared with 40% of A/A or A/G recipients. The CTLA-4 + 49 A/G SNP, which results in an amino acid substitution in the signal peptide of the protein, did not, however, affect intracellular expression or trafficking of CTLA-4 in T cells, nor soluble serum CTLA-4 concentrations of the liver transplant recipients. In conclusion, this pilot study suggests that liver transplant recipients homozygous for CTLA-4 + 49 G have a reduced risk of acute rejection. [source] Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 5 2003Lina Gu A gene encoding manganese peroxidase (mnp1) from Phanerochaetechrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P.chrysosporium fungal secretion signal, p,AMNP contains an ,-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and ,-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55,100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous MnII, CaII, and FeIII conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 °C. [source] Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytesBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009M. Kobayashi Summary Background, Epidermal keratinocytes are involved in the skin innate immunity and express toll-like receptors (TLRs) and other innate immune proteins. The epidermis is continuously exposed to pathogenic Gram-positive bacteria or fungi. However, few studies have examined the function and expression of innate immune proteins in keratinocytes. Histamine, which is well known for itch and allergy, is closely associated with innate immunity, but its influence on epidermal innate immunity is still unclear. Objectives, To clarify the expression of innate immune proteins in keratinocytes stimulated by ligand pathogen-associated molecules, and the function of histamine in this process. Methods, We investigated the effects of lipopeptide (MALP-2, 1,100 ng mL,1; ligand for TLR2), peptidoglycan (PGN, 0·02,2 ,g mL,1; ligand for NOD2) and ,-glucan (1,100 ,g mL,1; ligand for dectin-1) in the presence or absence of histamine on mRNA expression of TLR2, NOD2 and dectin-1 as well as human ,-defensin 2 by quantitative real-time polymerase chain reaction in cultured normal human epidermal keratinocytes. TLR2 expression was also examined at the cell surface and intracellularly, as determined by flow cytometry and confocal microscopy. The quantities of interleukin (IL)-1, and IL-8 produced by keratinocytes were measured using enzyme-linked immunosorbent assay. Results, At the mRNA level, TLR2 was enhanced by PGN but not by its ligand MALP-2 or by ,-glucan; NOD2 was easily induced by all three ligands; and dectin-1 was enhanced by its ligand ,-glucan. These enhanced expressions were further augmented by histamine at 1 ,g mL,1. While the surface expression of TLR2 was barely detectable by flow cytometry even after stimulation, the intracellular expression of TLR2 was apparently elevated by PGN and further promoted by histamine. A confocal microscopic analysis also revealed the enhanced expression of TLR2 in the cytoplasm. The expression of TLR2, NOD2 and dectin-1 was functional, as these pathogen-associated molecules induced the production of IL-1,, IL-8 and defensin, and again, histamine greatly enhanced this production. Conclusions, Our study demonstrated that the expression of functional innate immune receptors is augmented by the pathogen-associated molecules in a ligand-feed forward or nonrelated manner in keratinocytes, and histamine promotes their expression and the resultant production of cytokines and defensins. [source] Increased frequency of intracellular interleukin (IL)-13 and IL-10, but not IL-4, expressing CD4+ and CD8+ peripheral T cells of patients with atopic dermatitisBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2002M. Aleksza Summary Background A number of studies exist demonstrating the increased expression of type 2 cytokines and decreased capacity to produce interferon-, (IFN-,) in peripheral blood mononuclear cells (PBMCs) of patients with atopic dermatitis (AD). Objectives To clarify the results of recent studies concerning the role of interleukin (IL)-4 and IL-13 in PBMCs of AD patients, we analysed the activation status of lymphocyte subpopulations. Methods We measured the intracellular expression and serum levels of certain type 1 and type 2 cytokines, using cell surface and intracellular cytokine staining, flow cytometry and enzyme-linked immunosorbent assay techniques. Results The frequency of IL-10 and IL-13 producing CD4+ and CD8+ T cells was significantly higher in patients with AD, while the frequency of IFN-, secreting helper and cytotoxic T cells was significantly lower in patients with AD than in control subjects. The serum levels of IL-10 and IL-13 were also significantly increased. There were no significant differences observed between the experimental groups in the frequency of IL-4 producing CD4+ and CD8+ cells. Conclusions This study demonstrates a type 2 cytokine production in the CD4+ and CD8+ T cells of AD patients, which is characterized by an elevated IL-13, but not by IL-4 secretion, and by an increased level of the immunoregulatory IL-10, which can contribute to a decrease in IFN-, expression. [source] Local release of eosinophil peroxidase following segmental allergen provocation in asthmaCLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2003V. J. Erpenbeck Summary Background Eosinophil peroxidase (EPO) is an eosinophilic basic protein, which leads to increased permeability and damage of bronchial epithelial cells in asthma. Objective As little is known about its local expression and release in humans the intracellular expression in lung and peripheral eosinophils and the concentrations of EPO in bronchoalveolar lavage (BAL) fluid and serum was investigated in patients with asthma. Methods Twelve mild atopic asthmatic and nine control subjects underwent segmental sham and allergen challenge. EPO concentrations in BAL fluid and serum were determined by immunoassay and flow cytometry was used to determine the intracellular expression of EPO in BAL-derived and peripheral eosinophils. Results In asthmatic patients a large increase in BAL eosinophils , total cells: median 9.5 × 106 (range: 0.5 to 455.0 × 106); relative: 38% (1 to 91%) , was detectable 24 h following allergen challenge, but peripheral blood eosinophil counts did not change. Concentrations of EPO in BAL fluid increased from 1 µg/L (1.0 to 6.8 µg/L) to 42 µg/L (5.6 to 379.6 µg/L; P < 0.01) after allergen but not after saline challenge (1.5 µg/L; 1.0 to 21.9 µg/L), whereas in control subjects all measurements were below the detection limit. Serum concentrations of EPO increased slightly from 18.3 µg/L (3.0 to 56.8 µg/L) to 27 µg/L (3.8 to 133.9 µg/L; P < 0.05) 24 h after allergen challenge in asthmatic patients. Furthermore, the intracellular expression of EPO (measured as mean fluorescence intensity) was decreased in BAL eosinophils compared with blood eosinophils (mean fluorescence intensity 29 (7 to 71) vs. 48 (20 to 85); P < 0.01) after allergen challenge. Conclusion The finding of increased EPO concentrations in the BAL fluid and decreased intracellular EPO expression in pulmonary eosinophils of asthmatic patients reflects the allergen-triggered release of EPO into the bronchial space. [source] | |||||||||||||