Surface IgE on human basophils during histamine release.

The distribution of surface IgE on human basophils was studied using fluorescence microscopy and immunoferritin electronmicroscopy. Redistribution of the IgE was dose, time and temperature dependent and required divalent anti-IgE. Cells which can release histamine in vitro were indistinguishable in these respects from cells which cannot. The redistribution was unaffected by the presence or absence of Ca(++). No correlation between the conditions required for optimal histamine release and for redistribution was observed. At low doses, optimal histamine release occurred in the absence of, or before, redistribution. At higher doses redistribution occurred in the absence of histamine release. Antigen-induced histamine release was unaccompanied by gross redistribution of the surface IgE. Since both histamine release and redistribution require bridging of IgE on basophils it is concluded that only certain kinds of cross-linking of the IgE effectively stimulates these cells.

Mast cell clones: a model for the analysis of cellular maturation.

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.

Anaphylatoxin-induced histamine release with human leukocytes: studies of C3a leukocyte binding and histamine release.

Purified human C3a and synthetic COOH-terminal peptides of C3a, i.e., a pentapeptide, Leu-Gly-Leu-Ala-Arg (5R), and an octapeptide, Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg (8R) induced histamine release from human basophil granulocytes. On a molar basis, 5R was one-tenth and 8R was one-fifth as active as C3a in causing histamine release. It was found that 125I-C3a binds to whole leukocytes, interacting with both mononuclear cells and neutrophils and the binding was inhibited by preincubation of cells with unlabeled C3a, but not by C5a. 5R and 8R also inhibited the binding of 125I-C3a to the cells. However, on a molar basis, 2,000 times more 8R or 6,000 times more 5R is required for 50% inhibition of 125I-C3a binding as compared with native C3a. Autoradiography of cells using 125I-C3a and 125I-C5a showed preferential binding of 125I-C3a to eosinophils and basophils, whereas 125I-C5a binds primarily to neutrophils and eosinophils and to a lesser extent to basophils. The preferential binding of C3a and C5a to different cell types may herald significance related to their physiological functions.

Development of human mast cells from their progenitors.

Two types of human mast cells, which are morphologically similar to skin mast cells and lung mast cells, respectively, can be developed from pluripotent stem cells under different culture conditions. The major growth factor for mast-cell development is c-kit ligand, which induces mastocytosis in vivo. However, this cytokine is not sufficient for full maturation of the cells.

Fabrication of size-controlled polyimide nanoparticles.

Polyimide particles were fabricated through the two-steps imidization of poly(amic acid) particles prepared by using reprecipitation method. PAA and PI nanoparticles were all spherical, and the changes of particle size, its distribution, and morphology were not observed before and after the imidization. The preparation of PI nanoparticles size-controlled between ca. 20-500 nm was also achieved by changing the experimental conditions, temperature of the poor solvent, the composition of two kind of poor solvent, and PAA-NMP solution concentration.

Early experience with low-prime (99 ml) extracorporeal membrane oxygenation support in children.

Quick setup is mandatory for cardiopulmonary resuscitation using an extracorporeal membrane oxygenation (ECMO) assist device. Our conventional ECMO circuit for pediatric patients consists of a centrifugal pump (CX-HP) and membrane oxygenator (CX10H). Because of the large priming volume (260 ml), the circuit had to be primed with donor blood and required 30 minutes for setup. We started to use a low-prime ECMO with small centrifugal pump (HPM-15) and membrane oxygenator (MENOX Alpha Cube) for induction of ECMO beginning in 2000. The priming volume of this low-prime circuit is only 99 ml. The circuit can be primed without donor blood, even in the small patient, and requires only 10 minutes to set up. We review our experiences with cardiopulmonary resuscitation for sudden cardiopulmonary collapse in pediatric patients, including postcardiotomy patients. From 1997 to 2000, 23 patients underwent ECMO support with a conventional circuit (group A). From 2000 to 2004, we used low-prime circuit for induction of ECMO in 12 patients (group B). After the induction of ECMO with low-prime circuit, ECMO was converted to conventional heparin-bonded circuit for the longer support. The results suggested that the quick induction of ECMO with low-prime circuit has significant advantages in cardiopulmonary support in pediatric patients.

Arachidonic acid metabolism during antigen and ionophore activation of the mouse bone marrow derived mast cell.

This study has examined the metabolism of arachidonic acid in the mouse bone marrow-derived mast cell (BMMC) during immunologic and nonimmunologic activation. The predominant pools of endogenous arachidonate in the mast cells were found in ethanolamine (46%), choline (39%) and inositol (14%) containing glycerolipids. Initial studies established conditions where equilibrium labelling of these major phospholipids in the BMMC could be reached. Upon challenge, arachidonate was lost from all major phospholipid classes (phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol). There was a small but significant increase in the amount of label associated with phosphatidic acid during cell activation. Arachidonate was distributed among 1-acyl, 1-alkyl and 1-alk-1-enyl-linked subclasses of PC and PE. The rank order of loss of labelled arachidonate from the major PE and PC subclasses during antigen and ionophore activation was 1-alk-enyl-2-arachidonoyl-GPE greater than 1-acyl-2-arachidonoyl-GPC greater than 1-acyl-2-arachidonoyl-GPE greater than 1-alkyl-2-arachidonoyl-GPC. Labelled products released into the supernatant fluids and free arachidonic acid within the cell accounted for the bulk of arachidonate lost from phospholipids. Labelled products in the supernatant fluids were composed of LTB4, LTC4, PGD2 and free arachidonic acid. BMMC phospholipids were also labelled for 24 hr with [3H]choline, [3H]myoinositol or [14H]ethanolamine and labelled 2-lyso phospholipids were measured after cell activation. Radioactivity in lysophospholipids from PC, PE and PI increased significantly between 30 s and 2 min after antigen activation and then declined. Taken together, these studies suggest that arachidonate is mobilized predominantly from PE and in particular 1-alk-1-enyl-2-arachidonoyl-GPE by the direct removal of arachidonate from the sn-2 position of the molecule. Most of this arachidonate is then released from cells as eicosanoids or free fatty acid.

Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; distinction from mature basophilic leukocytes undergoing secretion in the same cultures.

The ability to culture human mast cells and, thus, to determine their ontogeny and possible relationships to other lineages has been facilitated by new studies using cocultures of cord blood cells with mouse fibroblasts and recombinant human or murine c-kit ligand-supplemented suspension cultures of cord blood cells. In this study, we examined c-kit ligand-supplemented cord blood cell suspension cultures designed so that the effects of growth factor source, individual cord sample, and culture time (3-17 weeks) on the developing mast cell lineage could be individually evaluated. We found that human mast cells, basophils, neutrophils, eosinophils, macrophages, megakaryocytes, and endothelial cells were present in these cultures. The numbers of mast cells and their granules increased with culture time; mature basophils, present in quantity in 3-week cultures, decreased in number and released granule contents with increased culture times. The mast cell lineage developed similarly, regardless of which factor preparation was added to cultures, but considerable variability existed among individual donors from whom cord bloods were obtained. Unlike the mature, crystal-containing mast cells that regularly developed in fibroblast cord blood cocultures (Furitsu et al. [1989] Proc. Natl. Acad. Sci. USA 86, 10039-10043), human mast cells failed to attain full maturity in the suspension cultures examined here, regardless of individual cord sample, added growth factor, or culture time. Furthermore, unlike cells of the basophil lineage in which granule content release was regularly observed, morphologic evidence of secretion from human mast cells was absent. Instead, these cells were actively undergoing granule building as determined by the increasing numbers of granules and filling of these containers over culture time. Crystal granules never developed, even at the maximum culture time of 17 weeks. We conclude that fibroblasts are necessary and sufficient for the differentiation and maturation of human mast cells in vitro from their agranular precursors in cord blood but that soluble c-kit ligand is not.

Ultrastructural identification of human mast cells resembling skin mast cells stimulated to develop in long-term human cord blood mononuclear cells cultured with 3T3 murine skin fibroblasts.

Human mast cells developed in vitro when cord blood mononuclear cells were cocultured for 3 months with 3T3 embryonic mouse skin fibroblasts. The metachromatic cells that arose in these cultures contained histamine, a functional Fc epsilon receptor and granule proteases (tryptase, chymase), and they were definitively identified by the ultrastructural demonstration of crystal granules. We present a detailed ultrastructural analysis of this newly available system for the reliable development of human mast cells in vitro and provide criteria for definitive identification of the mast cell and basophil lineages in humans.

Solubilization and initial biochemical characterization of an IgE-destroying enzyme on rat peritoneal mast cells.

Previous studies had demonstrated that incubation of IgE with purified rat mast cells can result in the time and cell concentration-dependent destruction of the ability of the IgE to be bound to specific receptors on rat basophil leukemia cells. The IgE-destroying activity, which has an extremely acid pH optimum, resisted attempts at solubilization using detergents. However, it was solubilized in good yield by use of chaotropic salts and especially KOCN. The soluble activity is stable to freezing and thawing, and to heating to 68 degrees C for 60 min. It is promptly destroyed upon boiling. IgE destruction was linear with time up to 20 min and a series of products, mol. wts 138,000, 92,500, 60,000 and 36,500, are formed during the reaction. No pH optimum for the reaction could be found because, as the pH was lowered below 4.0, the spontaneous destruction of IgE became too great. At pH 4.75 the apparent Km for the reaction was 0.55 microM and Vmax was 0.4 nmoles IgE/10(4) mast cell equivalents/min. IgE-destroying activity could be inhibited by heat-inactivated serum, and by relatively high concentrations of crude alpha 1-antitrypsin, aprotinin, lima bean and soybean trypsin inhibitors and by p-nitroguanidinobenzoate. A large number of other protease inhibitors were inactive.

C-kit ligand induction of immature neutrophils in cultures of human umbilical cord blood.

Human cord blood cells cultured in suspension with soluble c-kit ligand produce immature mast cells from their agranular precursors; cocultures of cord blood and mouse 3T3 fibroblasts produce fully mature human mast cells. We noted cells of the neutrophil lineage in the c-kit ligand-supplemented suspension cultures. Similar cultures were prepared from individual cord bloods with several sources of the c-kit ligand, including mouse fibroblast conditioned media, a partially purified mouse 3T3 fibroblast factor(s), recombinant human stem cell factor, recombinant murine mast cell growth factor, and were sampled sequentially for routine and cytochemical ultrastructural studies. These studies show that peroxidase-positive azurophilic granule-containing neutrophilic myelocytes develop in quantity from their agranular precursors in cord blood when the c-kit ligand is present, but little to no maturation to mature neutrophils with specific granules occurs. Specific granules were also absent in the neutrophil precursors. The effect of c-kit ligand in vitro on two cell lineages in man is similar--i.e., it permits the development of immature cells to differentiate from their agranular precursors in cord blood, but complete maturation to fully mature mast cells or neutrophils does not occur.

Charcot-Leyden crystal protein distribution in basophils and its absence in mast cells that differentiate from human umbilical cord blood precursor cells cultured in murine fibroblast culture supernatants or in recombinant human c-kit ligand.

Suspension cultures of human umbilical cord blood mononuclear cells supplemented with c-kit ligand-containing additives give rise to a mixture of cells belonging to several lineages. Among those that differentiate in quantity are mature basophils, immature mast cells, and neutrophilic myelocytes. We used an ultrastructural immunogold method to detect the Charcot-Leyden crystal (CLC) protein, an eosinophil- and basophil-specific protein, to study cells that were obtained at sequential times from 3 to 14 weeks in culture. Basophils (and eosinophils, which were present in smaller numbers) labeled for the CLC protein; mast cells did not. The labeled basophil subcellular sites included formed intragranular, cytoplasmic and nuclear CLCs, cytoplasmic particle-filled and homogeneously dense granules, cytoplasm, nucleus, plasma membrane, and cytoplasmic and Golgi area vesicles. Individual basophil ultrastructural phenotypes similar to those associated with stimulated release and recovery reactions showed the expected variations in the gold-labeled subcellular compartments. Macrophages also were labeled for CLC protein within endocytotic-lysosomal structures; neutrophilic myelocytes did not contain CLC protein. On the basis of findings reported here, the combined ultrastructural morphology and immunogold phenotyping of cells differentiating in c-kit ligand-supplemented cultures allows accurate lineage assignment of the developing cells.

Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro.

We examined the ultrastructural localization of (a) a secondary granule matrix protein--eosinophil peroxidase (EPO)--by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMs) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhIL-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMs, as well as identification of unicompartmental, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.

Induction of differentiation of the human leukemia cell line, KU812.

We treated KU812 cells from different passage levels with compounds which induce differentiation of other leukemic cell lines. Early passages contained small subpopulations of basophil and erythrocyte-like cells, but later passages consisted of more uniform cells with a myelomonocytic phenotype. When cultured with 1 nM actinomycin D, the cells became nonproliferative, developed pale cytoplasm and segmented nuclei, and lost nonspecific esterase activity. Thus, KU812 cells can be induced to mature to cells with a myelomonocytic phenotype. However, their expression of previously reported basophil characteristics diminishes with continued passage, and may be a capability of only a subpopulation of the cells.

Human eosinophils in vitro. An ultrastructural morphology primer.

An ultrastructural morphological primer of human eosinophils is presented. Mature and immature eosinophils, obtained from peripheral blood and bone marrow, as well as activated tissue eosinophils are all used to illustrate the various morphologies assumed by eosinophils in vivo. The various ultrastructural changes expressed by this cell lineage in vivo reflect the impact of differentiation, maturation, activation, secretion, and cell injury on morphology. Nearly all of the changes described in vivo are also evident in eosinophils arising in in vitro systems. We review published studies of these culture systems, which have been supplemented with various conditioned media containing naturally occurring growth factor(s) that are permissive (or not permissive) for eosinophils or with the recombinant growth factors, IL-5 or IL-3. These studies were helpful in the recognition of eosinophil-promoting, -sustaining and -activating properties of human IL-3 and IL-5. Moreover, mature and immature eosinophils were shown to release a granule matrix protein--eosinophil peroxidase (EPO)--by its transport in small cytoplasmic vesicles, a process termed piecemeal degranulation (PMD), accounting for the gradual emptying of granule contents in the absence of granule fusions to the plasma membrane. Also presented are eosinophil morphologies that occur in vitro in suspension cultures of human cord blood supplemented with the c-kit ligand from various sources. The wide variety of eosinophil subcellular changes in the c-kit ligand-supplemented cultures, like the changes of which eosinophils are capable in vivo, reflects the processes of differentiation, maturation, activation, secretion and cell injury. Presentation of this ultrastructural morphological primer of human eosinophils in vitro should enable investigators to recognize eosinophils in all of their diverse morphologic forms in cultures that contain differentiating and functioning members of other lineages, also present in c-kit ligand-supplemented cultures. These lineages include mast cells, basophils, neutrophils, monocytes, macrophages, megakaryocytes, and endothelial cells.

Artificial chordae for mitral valve reconstruction in children.

BACKGROUND: Congenital mitral regurgitation continues to present a challenge for cardiac surgeons because of the diversity of the anatomy of the congenitally malformed mitral valve. We undertook aggressive repair of the mitral valve with artificial chordae for reconstruction of the prolapsed anterior leaflet in some children. The short-term results are reported herein. METHODS: Three patients with isolated congenital mitral regurgitation underwent mitral valve repair with use of expanded polytetrafluoroethylene sutures as artificial chordae. RESULTS: There have been no late deaths and no valve-related complications. Serial follow-up echocardiographic examinations have not revealed any increase in the severity of mitral regurgitation with continuing patient growth up to 39 months after the operation. CONCLUSIONS: When combined with other conservative methods of mitral valve repair, chordal replacement with expanded polytetrafluoroethylene sutures in children undergoing mitral valve reconstruction produces good short-term results. We believe that it delays and possibly prevents the need for a mechanical prosthesis with its associated complications in this young patient population.

In vitro drug release from egg albumin microcapsules.

The in vitro release of phenacetin from microcapsules prepared using egg albumin as the membrane material was investigated. It was shown by scanning electron microscopy that the albumin microcapsules have nonsmooth surfaces. The amount of phenacetin released is proportional to the square root of time up to 50-70% drug release. Increases in the albumin concentration and 1-vinyl-2-pyrrolidinone polymer content in the aqueous phases used in the microcapsule preparation have an effect on matrix porosity and channel tortuosity in the matrix of albumin microcapsules. The in vitro release rate was found to decrease with increasing albumin concentration and 1-vinyl-2-pyrrolidinone polymer content in the aqueous phases. The in vitro release rate per unit area also decreased with decreasing capsule size.

Preparation of egg albumin microcapsules and microspheres.

Protein microspheres and microcapsules containing fine hydrophilic particles (spherical silica gel beads, iron hydrous oxidemodified nylon 12, or phenacetin) were prepared by using water-soluble egg albumin, which is heat-coagulable at 50-80 degrees. The size and size distribution of the microspheres and microcapsules formed and the microencapsulation percentage of fine particles were determined, and the factors affecting them were studied. Favorable conditions are suggested to obtain phenacetin-containing microcapsules in high yield.

Effects of acrylic acid polymer and its arrangement on drug release from a wax matrix.

The release of phenacetin from a wax matrix was improved by the addition of an acrylic acid polymer. Increasing the amount of polymer increased the release rate of phenacetin due to the formation of pores and channels in the matrix resulting from leaching of the polymer. The leaching was affected by the pH of the dissolution medium as well as by the methods used to prepare the granules. The polymer in ordered powder mixes behaved differently from that uniformly dispersed in a wax matrix. The drug appeared to be released by an approximate zero-order process at pH 7.5 from tablets containing the polymer.

Preparation of serum albumin microcapsules.

Simple coacervation of bovine serum albumin was studied to prepare biodegradable microcapsules. Three types of microcapsules, which differed in shape, were obtained by changing either core size or the bovine serum albumin concentrations of the coacervating systems. Mononuclear microcapsules were prepared by using spherical poly(acrylonitrile) beads as a core material.