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At least 649 records · Page 36Linked to original sources
PubMed2000

The situation of preimplantation genetic diagnosis in Germany: legal and ethical problems.

Preimplantation genetic diagnosis (PGD) has not been performed in Germany. Existing German laws do not explicitly permit or prohibit PGD. German law does forbid the wasting of totipotent embryonic cells, fertilizing oocyctes without transferring them in the same cycle, and the use of micromanipulations that later may harm embryonic development. German law also limits the number of oocytes allowed to be fertilized in each in vitro fertilization (IVF) cycle to three and prohibits research on embryos. The existence of these laws may impede but not prevent the development of PGD programmes in Germany. In contrast, the liberal regulation of pregnancy termination coupled with the development of ethical guidelines specific to PGD may facilitate the introduction of PGD in Germany. Because of these contradictions, legislation is required to clarify the legal status of PGD in Germany.

Abortion, Legal↗
PubMed2000

High levels of fetal erythroblasts and fetal extracellular DNA in the peripheral blood of a pregnant woman with idiopathic polyhydramnios: case report.

Abnormal amniotic fluid volume can be associated with increased maternal risk as well as perinatal morbidity and mortality. Polyhydramnios is often indicative of fetal, placental or maternal problems. In a large proportion of patients the aetiology of the disorder is unclear. Here we report on a case in which numerous fetal erythroblasts and large quantities of extracellular fetal DNA were found in the peripheral blood of a pregnant woman with idiopathic polyhydramnios bearing a male fetus. Following enrichment of erythroblasts by magnetic separation (MACS) and anti-CD71 antibodies, approximately 45-fold more erythroblasts were determined per ml peripheral maternal blood than in matched controls (231 versus 5). Single cell multiplex polymerase chain reaction (PCR) of individually micromanipulated erythroblasts showed that approximately 122 of these were of fetal origin. The concentration of extracellular fetal circulatory DNA in maternal plasma was determined by real-time quantitative PCR and shown to be almost double that of the control group (749.2 versus 404 fetal genome equivalents per ml maternal plasma). It can be speculated that the increased intrauterine pressure in polyhydramnios leads to an enhanced influx of fetal cells and free extracellular fetal DNA into the maternal circulation. This hypothesis will have to be tested with further cases.

Adult↗
PubMed2002

Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with postcolumn laser-induced fluorescence detection was used to individually detect 6.0, 1.0, 0.5, and 0.2 num diameter polystyrene microspheres and individually measure their electrophoretic mobility. The analysis of a nanoliter-size volume from a microsphere suspension results in an electropherogram characterized by several narrow spikes in a well-defined migration time window. Each spike is associated with one microsphere because, when one single microsphere is introduced into the capillary by micromanipulation, the electropherogram has only one spike in the same migration time window. The distributions of individual measurements resulting from an electropherogram were used to evaluate the reproducibility from run to run, observe the effect of sodium dodecyl sulfate (SDS) added to the running buffer, and to investigate the origin of electrophoretic dispersion. As expected from the interactions between microspheres and SDS, the addition of this surfactant to the running buffer narrowed the range and shifted the average electrophoretic mobility to more negative values. After evaluating common sources of broadening in capillary electrophoresis, electrophoretic dispersion was attributed to microsphere heterogeneity. Unlike electropherograms displaying Gaussian-like profiles, the two-dimensional representations of the individual measurements provide a new alternative to evaluate and study electrophoretic-related properties of microspheres.

Electrophoresis, Capillary↗
PubMed1997

B lymphocyte physiology: the beginning and the end.

Whereas lymphatic tissues were implicated as the chief sites for antibody production almost 100 years ago, three findings cemented the role of the plasma cell as the actual producer, namely the histological and tissue culture studies of Fagraeus, the beautiful immunofluorescent approach of Coons and his group, and the micromanipulation approach to the study of antibody formation by single cells introduced by Lederberg and myself. Proof that antibody-forming cells derived from B, rather than T, cells had to await the studies of Miller and Mitchell, with some minor technical contributions from myself. The physiology of the germinal centre also has a long history, and recently much interest has surrounded the relative roles of germinal centres as sites of somatic immunoglobulin V gene hypermutation and selection of high affinity B cells, versus the roles of extra-follicular proliferative foci as sources of the primary antibody response. Tolerance and self-antigens within the primary B lymphocyte repertoire is secured by clonal deletion within the bone marrow of those cells which are operationally the most threatening to the body, and a second level of functional impairment of B cell activity and life-span, capable of being induced centrally or peripherally, termed clonal anergy. As these concepts became progressively more refined through transgenic models of immunological tolerance, we turned our attention more towards tolerance within the secondary B lymphocyte repertoire. This is generated primarily inside germinal centres, where it appears that two quite separate mechanisms act as a bulwark against the possible creation of hypermutated anti-self cells. The first is that germinal centre activity and memory cell generation are dependent on antigen-specific, germinal centre-seeking CD4+ T cells, and if a putative anti-self mutant B cell gets no help because of T cell tolerance, it will not expand further. The second is a very specific mechanism confined to the germinal centre whereby antigen-specific B cells are especially sensitive to antigen-induced apoptosis if soluble, deaggregated antigen is presented to them before they reach the 'rescue' signal of follicular dendritic-cell-bound antigen. While some of the death in germinal centres is clearly apoptotic in nature, a further phenomenon observed electron microscopically relates to the formation of type B dark cells. It is not yet clear whether the DNA in this type of dying cell is cleaved. Transgenic expression of bcl-2 in germinal centre B lymphocytes confers incomplete protection from apoptosis caused by soluble antigen. The suggestion that at least some of this apoptosis is mediated via Fas-Fas ligand interactions is prompted by the observation that the apoptotic phenomenon is markedly reduced in lpr mice.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2001

Cytogenetic and molecular characterization of a patient with simultaneous B-cell chronic lymphocytic leukemia and peripheral T-cell lymphoma.

A patient is described who developed a peripheral T-cell lymphoma (PTCL) after a 6-year history of B-cell chronic lymphocytic leukemia (B-CLL). The progression of the T-cell disease spreading to pleura and skin terminated the course of the disease. A cytogenetic analysis performed six years after the first onset of the B-CLL showed the presence of two clones, one with trisomy 12 and another with inv(14)(q11q32.1) and trisomy 8. Combined immunophenotyping and fluorescence in situ hybridization demonstrated that only CD19+ cells contained a trisomy 12, whereas CD3+ cells contained a trisomy 8. Analyses of IgH and TCR rearrangements in single micromanipulated B- and T-cells lacked evidence for a clonal relation between B-CLL and PTCL cells. Based on our findings, we discuss the different hypotheses which might explain the development of simultaneous PTCL and B-CLL.

Aged↗
PubMed2001

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood.

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21.

Aneuploidy↗
PubMed1984

In vivo experimentation on rat incisor enamel organs through a surgical window.

Experimental agents administered systemically are costly and often toxic to animals. An in vivo technique has been developed whereby a surgical window in the alveolar bone allows selected areas of the rat incisor enamel organ and underlying enamel to be exposed to various drugs, radiolabeled molecules, and molecular weight markers. Sherman rats weighing 100 gm were anesthetized and the inferior surface of each hemimandible was surgically exposed. A slow-speed dental hand drill was used to drill a small hole through the alveolar bone overlying the secretion or maturation zones of the enamel organ. The wound was closed and during recovery the mechanical trauma to the underlying tissue moved away from the hole due to the continuous eruption of the tooth. Two to 5 days later the hole was reexposed and microinjections of 3H-proline, 125I-salmon calcitonin, vinblastine sulphate, and normal saline (as control) were administered through the hole with a micromanipulator and a microliter syringe. Radioautographic detection of 3H-proline incorporation in secretory ameloblasts and enamel at 10 minutes, 30 minutes, 1 hour, 4 hours, 1 day, and 2 days after microinjection was identical to that obtained previously by systemic injection. Two hours after microinjection of vinblastine sulphate the cellular response was again identical to that following systemic injection; 125I-salmon calcitonin (M.W. approximately 3,600D) was used as a molecular weight marker and was seen to diffuse into the enamel of the maturation zone at 10 minutes after microinjection. This study has demonstrated the feasibility of this new technique for experimentation on rat incisor enamel organs.

Alveolar Process↗
PubMed1995

Chromosomes take an active role in spindle assembly.

The assembly of a bipolar spindle is essential for the accurate segregation of replicated chromosomes during cell division. Do chromosomes rely solely on other cellular components to regulate the assembly of the bipolar spindle or are they masters of their own fate? In the Zhang and Nicklas(1) study reviewed here, micromanipulation techniques and video microscopy were used to demonstrate the different roles that chromosome arms, kinetochores and centrosomes play in bipolar spindle assembly.

Animals↗
PubMed2001

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.

Biomass↗
PubMed2002

Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production.

Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 microm. This, in turn, coincided with proportionately increased antibiotic production.

Bioreactors↗
PubMed1986

Microtubule-dependent reticulopodial motility: is there a role for actin?

We summarize our recent immunocytochemical characterization of the reticulopodial cytoskeleton of two allogromiid foraminifers and our pharmacologic dissection of its motility. The reticulopodial microtubule cytoskeleton stained with an antiserum to brain microtubule-associated protein 2. Polymeric actin was localized in the reticulopodia by rhodamine-phalloidin staining. Microtubule inhibitors reversibly inhibited all aspects of motility; cytochalasins induced altered morphology and disorganization of motility but did not inhibit pseudopodial movements or intracellular transport. Simultaneous application of KCN and salicylhydroxamic acid (an alternative oxidase inhibitor) rapidly blocked all movement, indicating that motility is dependent on metabolic energy and that an alternative oxidative pathway functions in allogromiids. Micromanipulation and laser microsurgical experiments revealed tension throughout the reticulopodium. Our results suggest that microtubules are active components of the reticulopodial motile machinery. Actin may mediate substrate adhesion, whole-cell locomotion, pseudopodial tension, and coordination of the microtubule-based motility.

Actins↗
PubMed1987

Role of cortical tension in fibroblast shape and movement.

In order to analyze the cellular mechanisms of shape formation, the shape of individual 3T3 cells was perturbed by micromanipulation resulting in the detachment and relaxation of a cellular extension and the bending of the extension to form an "elbow" at a variable angle beta. Finally, the tip of the extension was allowed to reattach to the substrate away from the cell. The cells reacted by drawing the extension tight. If beta less than 90 degrees, the elbow moved laterally for 8-15 min until the extension projected orthogonally at the cell surface. If beta greater than or equal to 90 degrees, the extension remained stationary. Finally, in all cases webs formed between attachment points in the perturbed area. If the tip of the extension was allowed to touch its own cell body, thus forming a loop, the cells invariably closed the loop. The paper interprets the cellular reaction as the result of cortical tension and suggests that it is a major factor in the formation of fibroblast shape and the expressions of fibroblast motility.

Animals↗
PubMed1989

Kinetics of granulocyte phagocytosis: rate limited by cytoplasmic viscosity and constrained by cell size.

Micromanipulation of yeast particles and blood granulocytes has been used to study the kinetics of single phagocytosis events. The ingestion process was quantitated by observation of sequential adhesion and encapsulation times. Both adherence and encapsulation times were found to increase greatly as the temperature was reduced below 37 degrees C; calcium in solution facilitated adhesion of the particle to the phagocyte but not encapsulation; both adhesion and encapsulation processes required a minimum level of plasma components (presumably complement). The general nature of these observations were confirmatory of previous studies, but this study is unique in that the specific time course of single particle ingestion was quantitated. It was immediately apparent that the phagocytosis process was 100% efficient above the threshold concentrations required for plasma and temperature, but variations in times from cell to cell indicated heterogeneity in the population. The total time for ingestion varied from as low as 2 sec/particle at 37 degrees C to above several min/particle below 15 degrees C. Encapsulation times for particles were normalized by estimates of particle surface areas to establish a specific time/unit area of particle surface: from 0.5 sec/10(-8) cm2 at 37 degrees C to greater than 8 sec/10(-8) cm2 at 15 degrees C. The temperature dependence of the encapsulation time correlated well with the temperature dependence of the "apparent" viscosity for granulocytes measured by micropipet aspiration. As such, the kinetic properties observed in these phagocytosis tests are consistent with a model that both assembly of the contractile system and the displacement of the surface by active contraction in phagocytosis are limited by viscous dissipation in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed2004

Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSC) are the routine techniques for fluorescent cell analysis. Recently, we developed a scanning fluorescent microscopy (SFM) technique. This study compares SFM to LSC (two slide-based cytometry, SBC, techniques) and FCM, in experimental and clinical settings. METHODS: For the relative cell-frequency determinations, HT29 colorectal cancer cells and Ficoll separated blood mononuclear cells (FSBMCs) were serially diluted (from 1:1 to 1:1,000) and measured by each of the three techniques. For the absolute cell number determinations (only for SBC) FSBMCs were smeared on slides, then HT29 cells were placed on the slide with a micromanipulator (5-50 cells). Tumor cells circulating in the peripheral blood were isolated by magnetic separation from clinical blood samples of colorectal cancer patients. All samples were double-stained by CD45 ECD and CAM5.2 FITC antibodies. For slides, TOTO-3 and Hoechst 33258 DNA dyes were applied as nuclear counter staining. RESULTS: In the relative cell frequency determinations, the correlations between the calculated value and measured values by SFM, LSC, and FCM were r(2) = 0.79, 0.62, and 0.84, respectively (for all P < 0.01). In the absolute cell frequency determinations, SFM and LSC correlated to a high degree (r(2) = 0.97; P < 0.01). CONCLUSIONS: SFM proved to be a reliable alternative method, providing results comparable to LSC and FCM. SBC proved to be more suitable for rare-cell detection than FCM. SFM with digital slides may prove an acceptable adaptation of conventional fluorescent microscopes in order to perform rare-cell detection.

Antiporters↗
PubMed1976

The relationship between storage and secretion of specific antibody by immune lymphoid cells: ultrastructural localization of anti-peroxidase antibodies in plaque-forming cells of the rabbit popliteal lymph node.

The ultrastructure of antibody-forming cells (AFC) has been studied in the lymph node cell population from rabbits locally immunized with horseradish peroxydase (PO) incorporated in complete Freund's adjuvant, and the kinetics of AFC development followed from day 7 to day 18 after one injection of PO. Identification of the AFC was done by local hemolysis assay, using carboxymethyl cellulose solidifying medium and PO-coated sheep erythrocytes. AFC were thereafter transferred by micromanipulation into a Beem capsule, fixed, treated by PO for fine ultrastructure detection of anti-PO antibodies, included, sectioned and studied by electron microscopy. It was found that the AFC were essentially of three categories: lymphocytes, proplasmacytes and plasmacytes, with (+), or without (-), intracellular antibody. The proportion of these categories varies with the time elapsed since the injection of antigen and with the plaque-forming activity of the population: lymphocytes (-) are relatively more numerous (over 30%) at the early stages of immunization (day 7). The number of plasmacytes increases with immunization. If most of them contain intracellular antibody at the early stages (up to day 9), the proportion of plasmacytes (+) decreases markedly afterwards. The cell type distribution is compatible with the idea that the lymphocytes are the precursors of plasmacytes, proplasmacytes being transitional forms, but no direct filiation scheme can actually be deduced from these experiments.

Animals↗
PubMed1984

Assignment of human natural killer (NK)-like cells to the T cell lineage. Single allospecific T cell clones lyse specific or NK-sensitive target cells via distinct recognition structures.

The aim of the present study was to define the cell lineage of mixed lymphocyte culture (MLC)-induced natural killer (NK) effector cells. Human MLC cells were plated under limiting microculture conditions in the presence of irradiated spleen cells and interleukin 2-containing supernatant. After 18 days, microcultures were scored for proliferation and for cytolytic activity against specific lymphoblasts and NK-sensitive K562 target cells. About 1 in 7 and 1 in 5 proliferating microcultures had specific or NK-like cytolytic activity, respectively. Moreover, several microcultures exhibited dual (specific and NK-like) cytolytic activity, even when they had been established at relatively low numbers of responding cells/well (0.5-0.25) to ensure a high probability of monoclonality. Direct evidence for the existence of cytolytic effector cells with dual activity was achieved by using clones derived from single MLC T cells by micromanipulation. Out of 26 cytolytic clones so derived, 16 exhibited specific cytolytic activity, whereas 22 lysed K562 target cells. More interestingly, 12 of these 26 clones were active against both types of target cells. Only one of these clones was able to lyse autologous or unrelated target cells. In contrast, all such clones lysed the NK-sensitive cell lines G11, MOLT-4, Raji, Daudi, Chang and T-24. Addition of saturating amounts of B9-4 monoclonal antibody in the lytic assays resulted in the inhibition of the specific cytolysis, but not the NK-like activity of clones with dual cytolytic activity. It thus appears that (a) alloreactive cytotoxic T lymphocytes can mediate both specific and NK-like cytolysis and (b) two independent recognition structures are involved in this dual activity.

Cell Differentiation↗
PubMed1985

The murine thymic nurse cell: an isolated thymic microenvironment.

The thymic nurse cell (TNC) consists of an epithelial cell enclosing lymphoid elements and is found in enzymic digests of the thymus. Although these structures have been implicated in the normal intrathymic development of T lymphocytes, little is known about the in situ structure of this unusual cell complex. In this study, various dyes were introduced into the intact thymus and their differential permeability was used to demonstrate that the TNC exists as a sealed structure in situ. The lymphocytes within the TNC were shown to be isolated from the general thymic environment. Preliminary studies on these lymphocytes and the physiology of their active release from individual, micromanipulated TNC in microcultures are reported.

Animals↗
PubMed1985

The limited immunocompetence of thymocytes within murine thymic nurse cells.

Thymic nurse cells, cortical epithelial cells enclosing 20-200 lymphocytes, were prepared from mouse thymus by enzyme digestion and repetitive sedimentation. Individual nurse cells were then isolated free of any exogenous thymocytes by micromanipulation, and the endogeneous thymocytes released from inside the nurse cells by a brief period of culture. The thymocytes from within individual nurse cells were tested, at the one cell/well level, for their capacity to proliferate in high cloning efficiency mitogen-stimulated limiting dilution cultures. The resultant clones were tested for their cytolytic capacity in a lectin-mediated isotype-release assay. Most intra-nurse cell thymocytes were unresponsive, like typical cortical thymocytes, but an average of 1/30, or around 2-6 lymphocytes/nurse cell, were able to proliferate in response to concanavalin A. The clones produced were of a relatively small size, similar to those characteristic of helper-lineage T cells. No cytolytic clones at all were obtained, despite stringent positive controls showing an efficient cytolytic response from known sources of cytolytic precursor cells. This finding disagrees with earlier studies on nurse cell lymphocytes, where there may have been a possibility of contamination with exogenous thymocytes. These results suggest either that the nurse cell represents a selective environment for helper-lineage T cell differentiation, or that further steps after the nurse cell stage are needed to produce mature cytolytic-lineage T cells.

Animals↗