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Heterogeneous cytokine production by acutely stimulated bronchoalveolar T lymphocytes in reovirus 1/Lang-infected mice.

While the in vitro properties of CD4(+) and CD8(+) cytokine-producing lymphocytes have been well studied, the in vivo cytokine production patterns and relative roles of CD4(+) and CD8(+) T lymphocytes during a primary in vivo immune response remain unclear. In this study, mice were inoculated intranasally with reovirus 1/L, and respiratory T lymphocyte populations were analyzed using multicolor flow cytometric analysis for the production of cytokine within and between classical type 1/type 2 patterns. Cytokine production observed in vivo following infection did not correlate with classical T cell cytokine expression patterns; instead, multiple types of lymphocyte populations that produced one of several possible cytokine combinations were present. Cytokine production by CD4(+) lymphocytes appears in the early and middle stages of the immune response, while CD8(+) lymphocytes produce more cytokine in the later stages. Early cytokine responses occurred predominantly in the whole lung and lung-associated lymph node populations. The complex patterns of cytokine expression seen in this study likely influence local cell-mediated immunity as well as the complex interaction of T cell subsets and the interaction of T cells with B cells which are necessary for the generation of cell-mediated and humoral immune responses required for effective broad-spectrum immunity.

Animals↗

Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation.

Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34+ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34+ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 x 10(4) to 0.65 x 10(4)/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamide-mobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34+ CD19+ (Leu 12) CD10+ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 +/- 16.9%, mean +/- 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34+ cells defined a CD45RA- CD71+ population containing 89 +/- 6.3% (n = 4) BFU-E and a CD45RA+ CD71+ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13+ CD45RA+ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34+ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34+ cells.

Adult↗

Peripheral human T lymphocyte maintenance of immune functional capacity and phenotypic characteristics following in vivo cocaine exposure.

The effects of cocaine exposure upon the host's immune response is equivocal since a variety of studies have generated conflicting conclusions, often as the result of differences between in vitro and/or animal models and the actual conditions experienced in humans who are acutely abusing this drug. To further address this issue, we have studied a group of patients who were positive for cocaine or cocaine metabolites and we evaluated a variety of functional parameters of T-lymphocytes and other peripheral lymphoid cell populations, as well as immunophenotypic characteristics of these cells. When compared to normal controls and patients who were negative for cocaine, we found that the cocaine-positive patients had T-cell functional assays which were essentially normal, with the exception of a slight depression in PHA stimulation. Likewise, the immunophenotype of the peripheral blood lymphocytic populations showed normal percentages and numbers of their T cell subsets (CD4, CD8), NK cells, and B cells. Multicolor flow cytometry analysis revealed no difference in T cell subpopulations positive for the "memory" marker, CD62L. No correlation could be established between levels of cocaine or cocaine metabolites and any phenotypic, demographic, or functional parameter. In summary, these results demonstrate that individuals acutely exposed to cocaine do not show markedly altered T cell function or fluctuations in phenotypically identified cell populations. These studies imply that acute cocaine exposure does not predispose individuals to grossly apparent immunosuppression. However, the possibility that subtle, transient, or more specific changes in the immune system may be incurred by use of cocaine, particularly with chronic exposure, remains to be determined.

Adult↗

Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.

Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence of numerous in situ DNA strand breaks is also an inherent feature of apoptotic cells. The aim of the present study was to differentially label DNA strand breaks in apoptotic cells vs photolysis-induced breaks in BrdUrd incorporating cells. This would allow one, by multicolor staining, to identify these respective cells in the same sample preparation. Toward this end, exponentially growing HL-60 cells were pulse labeled with BrdUrd and then were subjected to hyperthermia or treated with DNA topoisomerase I inhibitor camptothecin to induce apoptosis. DNA strand breaks in apoptotic cells were first labeled directly with fluorochrome-conjugated dUTP or dCTP, followed by dideoxynucleotide (to terminate chain elongation), in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The cells were subsequently exposed to UV light to photolyze DNA containing the incorporated BrdUrd. The photolysis-induced DNA strand breaks were, in turn, labeled with digoxygenin- or biotin-conjugated dUTP followed by digoxygenin antibody or avidin, respectively, conjugated with fluorochrome of another color. Alternatively, DNA strand breaks were labeled with BrdUTP which was then detected by FITC-conjugated anti-BrdUrd MoAb. Following counterstaining of cellular DNA with a fluorochrome of a third color it was possible to identify apoptotic cells, cells incorporating BrdUrd, and cells having no DNA strand breaks. Cell fluorescence was measured either by flow cytometry or with the microscope-based laser scanning cytometer. The SBIP approach also offers a possibility to study a colocalization of the immunocytochemically detectable cell constituents at the DNA replication points by microscopy. Using this approach the presence of the proliferating cell nuclear antigen at the DNA replication sites was revealed in MCF-7 breast carcinoma cells.

Antibodies, Monoclonal↗

Evidence against a looped structure of the inactive human X-chromosome territory.

Multicolor fluorescence in situ hybridization with a whole chromosome composite probe for the X-chromosome and microdissection probes for the Xp and Xq arms, as well as for the Xp terminal, Xq terminal, and X centromer specific subregional probes, was applied to three-dimensional (3D) preserved human female amniotic fluid cell nuclei. Confocal laser scanning microscopy and three-dimensional image analysis demonstrated distinctly separated Xp arm and Xq arm domains. 3D distance measurements revealed a high variability of intrachromosomal distances between Xpter, Xcen, and Xqter specific probes within both X territories. A 3D distance measurement error of +/- 70 nm was found in control experiments using quartz glass microspheres labeled with different fluorochromes. Our data argue against the hypothesis of Walker et al. (1991, Proc. Natl. Acad. Sci. USA 88, 6191-6195) that a looped structure of the inactive X territory is formed by tight telomere-telomere associations.

Female↗

Delivery of genes and fluorescent dyes into cells of the intact lens by particle bombardment.

The authors report the use of a particle bombardment technique to deliver exogenous genes and fluorescent dyes into living fibre cells in the intact lens. Gold particles were coated with plasmid DNA encoding green fluorescent protein (GFP) or the lipophilic fluorescent probes DiI and DiO. The particles were introduced into embryonic chicken or neonatal mouse lenses using a pressurized helium charge. A significant fraction of particles penetrated the capsule and came to rest in the superficial lens cortex. Over the range tested, varying the particle size or the pressure of the helium charge had little effect on the final distribution of particles within the lens. Particle bombardment was used initially to deliver DiI and DiO into the lens. Within a few hours, bombarded lenses exhibited multicolored membranous labelling of individual, elongating fibre cells. The particle bombardment technique was also used to introduce a plasmid encoding GFP. After overnight incubation, many fibre cells in the bow region expressed GFP. On close examination by confocal reflectance microscopy, each expressing cell was found to contain a gold particle lodged in its nucleus. The authors examined the fate of GFP-expressing fibre cells over a period of 1 week in organ culture. In the embryonic chicken lens, transfected fibres showed modest (approximately two-fold) elongation. In contrast, GFP-expressing mouse lens fibres underwent dramatic elongation, reaching the anterior and posterior sutures after 7 days in culture. These species differences may reflect the fact that mitosis continued at a near normal rate in the cultured mouse lens but declined precipitously in the cultured chicken lens. These results suggest that particle bombardment, in conjunction with appropriate cell culture conditions, may prove useful in visualizing the behaviour of differentiating fibre cells in the living intact lens in vitro.

Animals↗

The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2.

The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations viral RNAs and the site-selective editing of mammalian mRNAs of neural origin. The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2. Simultaneous multicolor FISH including lambda clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705.

Adenosine Deaminase↗

Visual demonstration of the organization of the human complement C4 and 21-hydroxylase genes by high-resolution fluorescence in situ hybridization.

We analyzed the gene organization in the complement component C4 and 21-hydroxylase (21OH) gene region of the human major histocompatibility complex using visual mapping of stretched DNA by multicolor fluorescence in situ hybridization (FISH). Normally, this region contains a duplicated 21OH-C4 gene cluster (21OHB-C4B-21OHA-C4A). Duplication and deletion of one or more copies of the 21OH-C4 gene unit are known to occur frequently. Biotin-labeled cDNA of the C4 gene and digoxigenin-labeled cDNA of the 21OH gene were hybridized to decondensed nuclei of peripheral blood lymphocytes obtained from individuals with various 21OH-C4 haplotypes. Hybridization signals of the C4 and 21OH probes were detected with fluorescein isothiocyanate (green) and rhodamine (red), respectively. Two linear green and red signal clusters were observed in each nucleus showing the normal haplotype. Gene duplication and deletion were visualized as addition and deletion of the signal cluster, respectively. The DNA types of the 21OH-C4 region determined by FISH were concordant with the results previously obtained by conventional molecular studies. Our high-resolution FISH technique is found to be useful for screening gene duplications and deletions.

Complement C4↗

A human chromosome 22 fosmid resource: mapping and analysis of 96 clones.

We have created a resource for chromosome 22 consisting of 96 unique, well-characterized Fosmids. The Fosmid vector permits efficient cloning of DNA fragments averaging 40 kb in a single-copy vector based on the F factor of Escherichia coli. We have found that Fosmid clones from human chromosome 22 show remarkable stability and are useful for a wide variety of applications in genome analysis. These 96 clones have been localized by FISH, using high-resolution fluorescent banding and multicolor mapping techniques, and their position on the chromosome was correlated with their content of a number of common repeated sequence elements. We identified a subset of clones likely to contain genes by restriction analysis using the enzymes NotI, MluI, SacII, and BssHII. This collection of cytogenetically anchored clones, representing nearly 7% of the chromosome, is of immediate value for detecting chromosomal rearrangements, for use in gene isolation, and as a framework for physical mapping.

Binding Sites↗

Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

Africa, Eastern↗

Spectral imaging and linear unmixing in light microscopy.

Fluorescence microscopy is an essential tool for modern biological research. The wide range of available fluorophores and labeling techniques allows the creation of increasingly complex multicolored samples. A reliable separation of the different fluorescence labels is required for analysis and quantitation, but it is complicated by the significant overlap of the emission spectra. This problem can be addressed on the acquisition and the processing side by the use of spectral imaging in conjunction with linear unmixing of the image data. This method allows the reliable separation of even strongly overlapping fluorescence signals and has become an important tool in colocalization and in FRET studies. In this chapter, the microscope techniques available for spectral imaging are presented and the theory of linear unmixing is explained. Possible limitations as well as approaches for image optimization are discussed to help to realize the full potential of this novel method. Biological applications that can be improved by spectral imaging and linear unmixing are presented.

Fluorescence Resonance Energy Transfer↗

Current radiodiagnostic concept of ankylosing spondylitis.

Nine radiodiagnostic rules are drawn from the X-rays of 2125 ankylosing spondylitis patients. The significance of early diagnosis of the 'multicolored' saroiliac X-ray is looked onto; the syndesmophyte, squaring-phenomenon, barrel-shaped vertebra, Romanus and Andersson lesions, and the ossification of ligaments are discussed. The changes at the apophyseal and the costovertebral joints, unimportant for early diagnosis, are explored.

Adult↗

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones.

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with "whole chromosome painting" probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter-->6p22 and a monosomy 8pter-->8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.

Abnormalities, Multiple↗

Topographic estimations by component spectroanalysis of two formazans of nitroblue tetrazolium in tissue sections.

A component-spectroanalysis technique was used to study the multicolor properties of histochemically stained tissue sections. We developed a method that makes it possible to obtain separately both the spectral patterns and spatial distributions of different color components in tissue sections. To illustrate the application of this technique, we examined the extinction spectrum of reduced nitroblue tetrazolium (NBT), which is used for the detection of dehydrogenase activity. Upon the reduction of NBT, mono- and diformazans are formed, and these exhibit over-lapping extinction spectra. When succinate dehydrogenase (SDH) activity in rat liver lobules was examined using NBT, monoformazan was found to be present at higher concentrations than diformazan and to have a uniform distribution, whereas the concentration of diformazan increased with a steep gradient between the center and periphery of lobules. In rat skeletal muscle fibers, diformazan was present at higher concentrations than monoformazan. The level of SDH activity was topographically represented by the hydrogen concentration calculated from the concentrations of the two formazans. This method is effective for separating multiple components such as mono- and diformazans in histochemical reactions.

Animals↗

Ordered mapping of three alpha satellite DNA subsets on human chromosome 22.

We report the physical order of three alphoid DNA subsets on human chromosome 22 determined by a combination of low- and high-resolution cytological mapping. Multicolor fluorescence in situ hybridization was performed on metaphase chromosomes, interphase nuclei and extended chromatin preparations. The results visually demonstrate the presence of three distinct alphoid DNA domains at the centromeric region of chromosome 22. Two domains appear adjacent by extended chromatin hybridization, while the third one is separated by DNA that does not hybridize with any of our probes. Our data demonstrate the applicability of interphase mapping for ordering alpha satellite DNA repeat arrays. However, in our experiments, the relationship between the extremities of repeat arrays could only be studied by extended chromatin experiments.

Chromosome Mapping↗

Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.

There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.

Animals↗

Spatial role-taking ability among bilingual and monolingual kindergarten children.

Fifty-seven children enrolled in a bilingual Spanish kindergarten program, assigned to appropriate language and age-related groups, were shown a graduated sequence of increasingly complex arrangements of multicolored blocks and were asked to judge how the original arrangement would look from the opposite and the side perspectives. A series of 2(younger vs. older) X 3(Spanish Monolingual vs. English Monolingual vs. Spanish-English Bilingual) ANOVAs for each of the types of responses (correct, incorrect, egocentric) showed a significant main effect for age on the incorrect answers. No differences associated with egocentrism were obtained. There was no relationship between age and success with simple and complex spatial tasks. As opposed to other studies that suggest certain cognitive advantages for young bilingual children, this study indicates no perceptible differences associated with being monolingual or bilingual at the ages of 5 and 6 for spatial tasks.

Child↗

Heidelberg Cytometry Symposium, 22-24 October 1992.

Lectures and presentations at the 1992 Heidelberg Cytometry Symposium reflected a rapidly growing research field with widespread activities that cover daily routine diagnosis as well as investigations at the molecular level and the diagnosis of genetic alterations. Both flow and image cytometry and their impact on quantitative cytology were backed and combined with new approaches (i.e. magnetic cell sorting) that enable the isolation of rare cells with high purity for cell biological analyses and thus pave the way for new research fields (i.e. arteriosclerosis). Their combination with the multicolor painting of gene sequences (fluorescent in situ hybridization) represents a further improvement of chromosome quantification and thus the analysis of the topology of cell nuclei. This year's meeting will be held from 21 to 23 October. Its topics will comprise chromosome painting, apoptosis, automated cytology, bio-imaging and proliferation. As usual, space is given to the presentation of new techniques and concepts. The deadline for abstracts is 30 June.

DNA, Neoplasm↗