scholarly journals Intracellular Localization of Interleukin-6 in Eosinophils From Atopic Asthmatics and Effects of Interferon γ

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2508-2516
Author(s):  
Paige Lacy ◽  
Francesca Levi-Schaffer ◽  
Salahaddin Mahmudi-Azer ◽  
Ben Bablitz ◽  
Stacey C. Hagen ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Human Eosinophil ◽  
Arylsulfatase B

Eosinophils, prominent cells in asthmatic inflammation, have been shown to synthesize, store, and release an array of up to 18 cytokines and growth factors, including interleukin-6 (IL-6). In this report, we show that IL-6 immunofluorescence localizes to the matrix of the crystalloid granule in peripheral blood eosinophils from atopic asthmatics using confocal laser scanning microscopy (CLSM). Granule localization of IL-6 was confirmed using dot-blot analysis and enzyme-linked immunosorbent assay (ELISA) on subcellular fractions of highly purified eosinophils produced from density centrifugation across a 0% to 45% Nycodenz gradient. IL-6 was found to coelute with eosinophil crystalloid granule marker proteins, including eosinophil peroxidase (EPO), major basic protein (MBP), arylsulfatase B, and β-hexosaminidase. Immunoreactivity to IL-6 colocalized with granule-associated IL-2 and IL-5 in subfractionated eosinophils. We also made the novel and compelling observation that interferon γ (IFNγ), a Th1-type cytokine, stimulated an early elevation in eosinophil IL-6 immunoreactivity. A 2.5-fold enhancement of IL-6 immunoreactivity in eosinophil granules was observed within 10 minutes of IFNγ treatment (500 U/mL), as determined by subcellular fractionation and CLSM. These findings suggest that IFNγ has short-term effects on human eosinophil function and imply that a physiologic role exists for Th1-type cytokine modulation of Th2-type responses in these cells.

scholarly journals Intracellular Localization of Interleukin-6 in Eosinophils From Atopic Asthmatics and Effects of Interferon γ

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2508-2516 ◽  
Author(s):  
Paige Lacy ◽  
Francesca Levi-Schaffer ◽  
Salahaddin Mahmudi-Azer ◽  
Ben Bablitz ◽  
Stacey C. Hagen ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Human Eosinophil ◽  
Arylsulfatase B

Abstract Eosinophils, prominent cells in asthmatic inflammation, have been shown to synthesize, store, and release an array of up to 18 cytokines and growth factors, including interleukin-6 (IL-6). In this report, we show that IL-6 immunofluorescence localizes to the matrix of the crystalloid granule in peripheral blood eosinophils from atopic asthmatics using confocal laser scanning microscopy (CLSM). Granule localization of IL-6 was confirmed using dot-blot analysis and enzyme-linked immunosorbent assay (ELISA) on subcellular fractions of highly purified eosinophils produced from density centrifugation across a 0% to 45% Nycodenz gradient. IL-6 was found to coelute with eosinophil crystalloid granule marker proteins, including eosinophil peroxidase (EPO), major basic protein (MBP), arylsulfatase B, and β-hexosaminidase. Immunoreactivity to IL-6 colocalized with granule-associated IL-2 and IL-5 in subfractionated eosinophils. We also made the novel and compelling observation that interferon γ (IFNγ), a Th1-type cytokine, stimulated an early elevation in eosinophil IL-6 immunoreactivity. A 2.5-fold enhancement of IL-6 immunoreactivity in eosinophil granules was observed within 10 minutes of IFNγ treatment (500 U/mL), as determined by subcellular fractionation and CLSM. These findings suggest that IFNγ has short-term effects on human eosinophil function and imply that a physiologic role exists for Th1-type cytokine modulation of Th2-type responses in these cells.

Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Paige Lacy ◽  
Salahaddin Mahmudi-Azer ◽  
Ben Bablitz ◽  
Stacey C. Hagen ◽  
Juan R. Velazquez ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Human Peripheral Blood ◽  
Subcellular Fractionation ◽  
Secretory Vesicle ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf ◽  
Ifn Γ

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-γ (IFN-γ) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-γ to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL per 2 × 106 cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-γ stimulation. This effect was not observed in eosinophils treated with IFN-, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-γ. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

Cell-cell cooperation at the T helper cell/mast cell immunological synapse

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 4979-4988 ◽  
Author(s):  
Nicolas Gaudenzio ◽  
Nicolas Espagnolle ◽  
Lennart T. Mars ◽  
Roland Liblau ◽  
Salvatore Valitutti ◽  
...  
Keyword(s):  
T Cells ◽  
Mast Cells ◽  
Cd4 T Cells ◽  
Laser Scanning ◽  
Class Ii ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Interleukin 4 ◽  
Confocal Laser ◽  
Peritoneal Mast Cells

Abstract It has been suggested that mast cells might serve, under certain circumstances, as antigen-presenting cells (APCs) for T cells. However, whether cognate interactions between mast cells and class II–restricted CD4+ T cells actually occur is still an open question. We addressed this question by using peritoneal cell–derived mast cells (PCMCs) and freshly isolated peritoneal mast cells as APC models. Our results show that in vitro treatment of PCMCs with interferon-γ and interleukin-4 induced surface expression of mature major histocompatibility complex class II molecules and CD86. When interferon-γ/interleukin-4–primed PCMCs were used as APCs for CD4+ T cells, they induced activation of effector T cells but not of their naive counterparts as evidenced by CD69 up-regulation, proliferation, and cytokine production. Confocal laser scanning microscopy showed that CD4+ T cells formed immunological synapses and polarized their secretory machinery toward both antigen-loaded PCMCs and freshly isolated peritoneal mast cells. Finally, on cognate interaction with CD4+ T cells, mast cells lowered their threshold of activation via FcϵRI. Our results show that mast cells can establish cognate interactions with class II–restricted helper T cells, implying that they can actually serve as resident APCs in inflamed tissues.

scholarly journals Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus

2011 ◽  
Vol 18 (9) ◽  
pp. 1568-1576 ◽  
Author(s):  
Ajay P. Nayak ◽  
Brett J. Green ◽  
Erika Janotka ◽  
Justin M. Hettick ◽  
Sherri Friend ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Laser Scanning ◽  
Room Temperature ◽  
Aspergillus Terreus ◽  
Leucine Aminopeptidase ◽  
Dipeptidyl Peptidase ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type

ABSTRACTAspergillus terreushas been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasiveA. terreusdisease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived fromA. terreusculture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific forA. terreus. HEA were detected in conidia, hyphae, and in CSN ofA. terreus. HEA were expressed in high levels in the hyphae during early stages ofA. terreusgrowth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

scholarly journals Improved Bioassay of Xylella fastidiosa Using Nicotiana tabacum Cultivar SR1

Plant Disease ◽  
2008 ◽  
Vol 92 (1) ◽  
pp. 14-20 ◽  
Author(s):  
M. Francis ◽  
E. L. Civerolo ◽  
G. Bruening
Keyword(s):  
Nicotiana Tabacum ◽  
Xanthomonas Campestris ◽  
Laser Scanning ◽  
Xylella Fastidiosa ◽  
Quantitative Polymerase Chain Reaction ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Inoculation ◽  
Leaf Scorch

Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fastidiosa cells by transmission and scanning electron microscopy, as well as by confocal laser scanning microscopy, in the xylem cells of inoculated plants. The pathogenicity of selected reisolated strains was confirmed by inoculation of grape plants in the greenhouse. The average levels of X. fastidiosa cells/g of tissue, estimated by QPCR, were higher for PD strains than for ALSD strains and reflected the relative titers of these strains in economic hosts. No symptoms were observed and bacteria were not detected in untreated tobacco or in tobacco inoculated with Xanthomonas campestris pv. campestris or water. Symptoms induced by Xylella fastidiosa in this bioassay were fully expressed within 2 months following inoculation. The described bioassay, under optimized environmental conditions, provides a useful system for studying X. fastidiosa strains (e.g., confirmation of pathogenicity and differentiation of PD and ALSD pathotypes) and for investigating X. fastidiosa–host interactions. N. tabacum cv. SR1 tobacco was a better bioassay host for X. fastidiosa than N. tabacum cvs. Havana, RP1, and TNN described previously.

BIOCONJUGATED QUANTUM DOTS BASED RAPID DETECTION OF PATHOGENIC BACTERIA FROM WATER SAMPLES

2011 ◽  
Vol 10 (01n02) ◽  
pp. 199-203 ◽  
Author(s):  
RICHA JACKERAY ◽  
GURPAL SINGH ◽  
SWATI JAIN ◽  
ZAINUL ABID CKV ◽  
HARPAL SINGH ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Salmonella Typhi ◽  
Laser Scanning Microscopy ◽  
Fluorescent Label ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Core Shell ◽  
Exchange Method

A sensitive and rapid method for the detection of pathogenic bacteria (Salmonella typhi) in water sample was developed using core-shell CdSe / ZnS quantum dots (QDs) as fluorescence label. Surface-functionalized core-shell quantum dots were synthesized by successive ion layer adsorption and reaction (SILAR) technique and were made hydrophilic by ligand exchange method. Developed hydrophobic and hydrophilic QDs were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and spectrofluorimetry. Carboxy- terminated QDs were conjugated with bacteria-specific antibodies (S. typhi-specific IgG) for the preparation of photostable fluorescent label and were characterized by various techniques like spectrofluorimetry and enzyme-linked immunosorbent assay (ELISA) for their photoluminescence and successful bioconjugation. Antibody (Ab)-conjugated QDs were incubated with bacteria-contaminated water for S. typhi detection. Microscopic images and spectral profile of bacteria–Ab conjugated QDs complex were recorded by confocal laser scanning microscopy (CLSM). A sensitivity of 103 organisms/mL of targeted bacteria (S. typhi) could be attained in a period of about 2 h.

scholarly journals Plant Host Range and Leafhopper Transmission of Maize fine streak virus

2010 ◽  
Vol 100 (11) ◽  
pp. 1138-1145 ◽  
Author(s):  
Jane C. Todd ◽  
El-Desouky Ammar ◽  
Margaret G. Redinbaugh ◽  
Casey Hoy ◽  
Saskia A. Hogenhout
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Streak Virus ◽  
Viral Titer ◽  
Plant Host ◽  
Acquisition Access Period ◽  
Access Period ◽  
Maize Fine Streak Virus

Maize fine streak virus (MFSV), an emerging Rhabdovirus sp. in the genus Nucleorhabdovirus, is persistently transmitted by the black-faced leafhopper, Graminella nigrifrons (Forbes). MFSV was transmitted to maize, wheat, oat, rye, barley, foxtail, annual ryegrass, and quackgrass by G. nigrifrons. Parameters affecting efficiency of MFSV acquisition (infection) and transmission (inoculation) to maize were evaluated using single-leafhopper inoculations and enzyme-linked immunosorbent assay. MFSV was detected in ≈20% of leafhoppers that fed on infected plants but <10% of insects transmitted the virus. Nymphs became infected earlier and supported higher viral titers than adults but developmental stage at aquisition did not affect the rate of MFSV transmission. Viral titer and transmission also increased with longer post-first access to diseased periods (PADPs) (the sum of the intervals from the beginning of the acquisition access period to the end of the inoculation access period). Length of the acquisition access period was more important for virus accumulation in adults, whereas length of the interval between acquisition access and inoculation access was more important in nymphs. A threshold viral titer was needed for transmission but no transmission occurred, irrespective of titer, with a PADP of <4 weeks. MFSV was first detected by immunofluorescence confocal laser scanning microscopy at 2-week PADPs in midgut cells, hemocytes, and neural tissues; 3-week PADPs in tracheal cells; and 4-week PADPs in salivary glands, coinciding with the time of transmission to plants.

scholarly journals Three-dimensional Imaging of the Intracellular Localization of Growth Hormone and Prolactin and Their mRNA Using Nanocrystal (Quantum Dot) and Confocal Laser Scanning Microscopy Techniques

2005 ◽  
Vol 53 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Akira Matsuno ◽  
Johbu Itoh ◽  
Susumu Takekoshi ◽  
Tadashi Nagashima ◽  
R. Yoshiyuki Osamura
Keyword(s):  
Growth Hormone ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Semiconductor Nanocrystals ◽  
Intracellular Localization ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

Semiconductor nanocrystals (Quantum dots, Qdots) have recently been used in biological research, because they do not fade on exposure to light, and they enable us to obtain multicolor imaging because of a narrow emission peak that can be excited via a single wavelength of light. There have been no reports of simultaneous localization of mRNA and protein using Qdots. We successfully applied these advantages of Qdot and confocal laser scanning microscopy (CLSM) to three-dimensional images of the intracellular localization of growth hormone and prolactin and to their mRNA. In situ hybridization and immunohistochemistry using Qdots combined with CLSM can optimally illustrate the relationship between protein and mRNA simultaneously in three dimensions. Such an approach enables us to visualize functional images of proteins in relation with mRNA synthesis and localization.

scholarly journals Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1

2011 ◽  
Vol 92 (7) ◽  
pp. 1519-1531 ◽  
Author(s):  
Sabine Feichtinger ◽  
Thomas Stamminger ◽  
Regina Müller ◽  
Laura Graf ◽  
Bert Klebl ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cdk Inhibitor ◽  
Cyclin T1 ◽  
Regulatory Impact ◽  
Transcription Complexes

Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269–487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69–cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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