Effect of colchicine on rat mast cells.

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.

The in vitro differentiation of mast cells. Cultures of cells from immunized mouse lymph nodes and thoracic duct lymph on fibroblast monolayers.

When cells from lymph nodes or thoracic duct of mice hyperimmunized with protein antigens are cultivated on embryo monolayers in the presence of the antigen, numerous clones of mast cells appear. The histochemical and ultrastructural characteristics of the cells permit their identification as mast cells and distinguish them from the phagocytic histiocytes that usually arise in abundance in similar cultures from unimmunized mouse cells or from immunized mouse cells cultured in the absence of the antigen. Only a few colonies of mast cells appeared in the latter cultures. The basis for the induction of mast cell differentiation is not known.

Aggregation and transformation of rat lymphocytes on rat embryo monolayers.

Lymphocytes from Sprague-Dawley rats have been cultured on monolayers of embryo-derived fibroblasts from the same outbred strain. Under these conditions the lymphocytes form aggregates, and transformation of small lymphocytes to lymphoid blast cells occurs within these aggregates. Transformation is characterized cytologically by enlargement of the nucleus, dispersion of nuclear chromatin, and the appearance of a prominent nucleolus. The principal cytoplasmic changes are an increase in cytoplasmic volume, a marked increase in number of ribosomes, and a clustering of ribosomes. These changes parallel those seen in the transformation of lymphocytes caused by a variety of treatments. One apparent difference is the paucity of lysosomes and lipid inclusions in the lymphocytes that transform on the monolayer.

Inhibition of mast cell histamine secretion by N-substituteed derivatives of phosphatidylserine.

The structural basis for the highly specific action of phosphatidylserine in enhancing mast cell histamine secretion induced by concanavalin A was investigated by studying the activities of three N-substituted derivatives: N-acetyl phosphatidylserine, N-1-dimethylaminonaphthalene-5-sulfonly phosphatidylserine, and N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylserine. None of the derivatives was capable of activating concanavalin A-induced histamine secretion at concentrations two to three times that required for maximal activation by phosphatidylserine. Instead, the derivatives were found to inhibit the secretory response of mast cells to the calcium ionophore A23187 as well as to concanavalin A. The inhibition was noncytotoxic, partially reversible by washing, and associated with binding of N-substituted phosphatidylserine to the mast cell.

Secretion from rat basophilic leukaemia cells induced by calcium ionophores. Effect of pH and metabolic inhibition.

Previous experiments on the functional properties of rat basophilic leukaemia cells showed a major anomaly when compared to normal mast cells: though IgE-mediated secretion was dependent on external Ca2+ with both types of cells, substantial non-cytotoxic release with ionophore A23187 could be demonstrated with the normal cells but not with the tumour cells. We now show that when the pH of the incubation medium is increased to 8 it is possible to obtain excellent Ca-dependent, non-cytotoxic secretion from tumour basophils with the ionophores A23187 and ionomycin. These results provide further evidence that secretion from the tumour cells occurs via a mechanism similar to that used by normal mast cells and basophils. Experiments with metabolically inhibited tumour cells suggest that their unusual sensitivity to the cytotoxic effects of Ca2+ ionophores may be related to their ability to sequester intracellular calcium. Changes in the conditions of cell culture appeared to produce substantial and at least partially reversible changes in responsiveness to IgE-mediated triggering and ionophores.

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C.

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.

Mast cell secretion: membrane events.

Stimulation of secretion with A23187 circumvents the usual mechanism of stimulation of secretion by direct mediation of an increase in cytoplasmic Ca++ and thereby permits study of the terminal components of the secretory process in which granules are externalized by membrane fusion events. Two alterations in the plasma membrane precede fusion: the formation of bulges and the aggregation of intramembranous particles. These changes require a permissive level of ATP and are sensitive to reagents that bind to intracellular protein sulfhydryl groups. They seem not to be attributable to a direct effect of Ca++ on membrane phospholipids. The cell components responsive to Ca++ and responsible for the alterations in the membrane are not known; neither microtubules nor actin filaments seem to qualify.

Protein malnutrition: effect on rat peritoneal mast cell number, histamine content, and IgE receptors.

To examine the effects of protein malnutrition on mast cells, rats were fed a protein-deficient diet (0.5% protein ad libitum) or normal diet (27% protein ad libitum or pair fed) for 16, 21, 27, or 57 d. Male rats in the different groups showed no significant differences in mast cell number or histamine content per mast cell. IgE binding sites as measured by flow cytometry were decreased in rats on the deficient diet. Even after stripping receptors of endogenous IgE and then labeling with fluorescent IgE, the difference remained, thus confirming the lower number of mast cell IgE receptors in rats maintained on the protein-deficient diet.

Abnormal mast cell granules in the beige (Chédiak-Higashi syndrome) mouse.

Mast cells of beige (C57BL/6J) (bg-j/bg-j) mice were examined histochemically and ultrastructurally. Mast cell granules in the beige mice were markedly enlarged and irregular in shape. Granule contents stained uniformly with acidified toluidine blue, but with ruthenium red and Alcian Blue-safranin, two components were evident. The rims of the abnormal granules stained with ruthenium red and with Alcian Blue; the centers of the granules were clear with ruthenium red and stained with safranin. Mast cell granules thus represent another abnormal organelle in the Chédiak-Higashi syndrome.

Mast cell granule formation in the beige mouse.

The formation of mast cell granules was studied in the beige mouse utilizing histochemistry and electron microscopy. The time and sequence of appearance of heparin, histamine and the chymotrypsin-like protease were normal. By electron microscopy, the initial formation of progranules and subsequent aggregation was normal, but the granules from early stages were abnormally large. Reorganization of intermediate granule forms to homogeneous mature granules was delayed. Late fusions of intermediate and/or mature granules were not observed. Our findings indicate that the defect lies in the excessive initial fusion of progranules rather than in continued formation of new progranules or in fusion of mature granules with one another.

The presence of mast cell granules in rat parotid secretory granule preparations.

Rat parotid secretory granule preparations contain, in addition to the acinar secretory granules, a second type of granule. Whereas the acinar granules lyse under hypotonic conditions, this second type of granule does not, thus providing a means for obtaining a fraction sufficiently enriched in these granules to allow for their characterization. In the present study, these granules are shown to possess demonstrable chymotrypsin-like enzyme activity. In the intact rat parotid, such activity is shown by histochemical methods to be present in the numerous mast cells residing in the connective tissue stroma, but no such activity exists in any of the parenchymal cells. On the basis of their electron microscopic appearance, enzyme activity, and physical characteristics it is concluded that the second type of granule present in rat parotid secretory granule preparations originates from stromal mast cells rather than from parenchymal cells.

Effect on mast cell histamine of inhibiting histamine formation in vivo with alpha-fluoromethylhistidine.

An irreversible inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine (FMH), was used to inhibit histamine formation by mast cells in vivo. Even at doses of FMH sufficient to reduce histamine formation more than 95%, the ability of mast cells to synthesize histamine recovered rapidly. It was possible, however, to sustain levels of histamine-forming activity below 10% of normal with continuous administration of FMH from subcutaneously implanted osmotic pumps. Administration of FMH under these conditions did not deplete significantly mast cell histamine but did prevent the increase in total mast cell histamine that occurs over 14 days and also prevented the reconstitution of mast cell histamine stores after depletion by treatment with polymyxin B.

Histidine uptake by isolated rat peritoneal mast cells. Effect of inhibition of histidine decarboxylase by alpha-fluoromethylhistidine.

Preincubation with (S)-alpha-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, was found to markedly reduce, but not eliminate, the uptake of [3H]histidine by rat peritoneal mast cells. The Vmax for histidine transport for cells in which decarboxylation of histidine had been completely inhibited was 11.9 pmoles per min per 10(6) cells, compared to a Vmax of 18.9 pmoles per min per 10(6) cells in the presence of active mast cell histidine decarboxylase. The Km of uptake was 139 microM in the presence of alpha-fluoromethylhistidine, several times higher than the Km of 44.0 microM in the uninhibited cell. alpha-Fluoromethylhistidine did not inhibit mast cell uptake of phenylalanine, a competitive inhibitor of histidine uptake but not a substrate for histidine decarboxylase; nor did it inhibit the uptake of histidine by non-mast cells, which lack histidine decarboxylase. Levels of intracellular [3H]histidine in mast cells were similar in the presence and absence of the decarboxylase inhibitor. Based on these observations, we propose that intracellular decarboxylation of histidine in the mast cell serves to specifically enhance the uptake of histidine by the relatively non-specific amino acid transporter present in the plasma membrane of the cell.

The importance of biventricular failure in patients with postoperative cardiogenic shock.

To evaluate the importance of severe biventricular failure in patients with postcardiotomy ventricular failure, we analyzed the data from 30 patients treated with ventricular assist devices (VADs) over a five-year period. All patients had profound postoperative ventricular failure refractory to drugs and an intraaortic balloon (IAB). Evaluation of preoperative ventricular function did not allow prediction of which patients would require VADs. However, the development of perioperative myocardial infarction was an important determinant of the need for postoperative support with a VAD. Twenty patients received only a left VAD (LVAD). Four of them had isolated left ventricular failure; 3 were weaned, and 2 survived. None of the 16 patients with biventricular failure who received only an LVAD were weaned. Ten other patients with biventricular failure received biventricular support, either with a right VAD and IAB, or with two VADs. Of these 10 patients, 5 were weaned and 3 survived. Considering all 26 patients with biventricular failure, those receiving biventricular mechanical support (10) had a better chance (p less than 0.025) of being weaned (5/10) and surviving (3/10) than those who received only an LVAD (0/16). We conclude that biventricular failure is common in patients with postcardiotomy ventricular failure and is often the result of perioperative infarction. While patients with isolated left ventricular failure did well with an LVAD only, those with biventricular failure required biventricular mechanical support for survival.

Etiology of intestinal damage in gastroschisis. III: Morphometric analysis of the smooth muscle and submucosa.

The response of intestinal smooth muscle to injury may explain some of the motility derangement observed in infants with gastroschisis. An experimental model of gastroschisis was created and a detailed analysis of the intestinal muscle layer was undertaken to study this response. An abdominal wall defect and evisceration of the bowel were carried out in fetal lambs at 80 days' gestation (full term, 145 days), with delivery at 100 days or 135 days. Smooth muscle cell size and number were determined by detailed morphometric analysis, proliferative rate was determined using proliferating cell nuclear antigen staining, and collagen content was determined by morphometry after Verhoeff van Gieson staining. Compared with controls, there was a significant increase in cell number (hyperplasia) in the gastroschisis animals at 100 days and an increase in size (hypertrophy) at 135 days. The proliferation rate of smooth muscle was significantly lower and the submucosal collagen thickness was significantly greater in the gastroschisis animals during both periods. These data suggest that gastroschisis is characterised by initial hyperplasia, with subsequent diminution in smooth muscle proliferation. The hypertrophy may reflect a response to injury in which cell growth instead of proliferation occurs. The persistent elevation in collagen throughout gestation in animals with gastroschisis may be a reflection of this hyperplastic response in the smooth muscle cells and an important factor in the bowel-wall thickening. This deranged pattern of growth may lead to the clinical problems observed in human infants with this disease.

Porphyrin content of the cysticercus of Taenia solium.

The strong red fluorescence of the cysticercus of Taenia solium depends on the presence of several porphyrins in the vesicular fluid of the parasite: probably protoporphyrin IX, coproporphyin I or III, and 2 decarboxylated porphyrins intermediate between uroporphyrin and coproporphyrin. Cyst porphyrins associated to form conglomerates of high molecular weight that dissociated in acid solutions and were not antigenic themselves nor associated with antigenic molecules. An appreciable fraction of the porphyrins was capable of undergoing oxidation and reduction, indicating that some of the porphyrins were complexed with metal ions. The metabolic basis for the accumulation of porphyrins is unknown. Preliminary results suggest that conditions deleterious to the cysticercus cause release of porphyrins so that the appearance of porphyrins in the cerebrospinal fluid of neurocysticercotic patients may prove useful in monitoring therapeutic attacks on the parasite.

Determination of mean particle volume, a Monte Carlo simulation.

Either length measurements or area measurements may be made on a sample of profiles for the purpose of estimating the mean volume of a population of convex particles. Diameters of spheres, caliper diameters of ellipsoids and intercept lengths are available length measurements. Profile areas can be evaluated by planimetry or point counting. Either all the available profiles in random sections or point sampled profiles can be utilized. We have applied a Monte Carlo simulation to compare several of the stereologic methods for the estimation of the mean volumes of spheres and ellipsoids. Populations of spherical, prolate ellipsoidal and oblate ellipsoidal particles were subjected to random sectioning and measurement. Diameter, point sampled intercept length, area and point sampled area were measured in the case of the spherical particles. With the ellipsoids, the same measurement excepting diameters were performed. The measurements were converted to volumes by the appropriate equations, and the means, the standard deviations of the means and the 95% confidence intervals were determined for increasing sample sizes. All the methods provide estimates that converge on their theoretical mean volumes. The area measurements and particularly the point sampled area measurement show some advantage over the length measurements, but differences among the methods are small, not entirely consistent over the different cases and unlikely to be significant in most real applications.

Loss of the xeroderma pigmentosum group B protein binding site impairs p210 BCR/ABL1 leukemogenic activity.

Previous studies have demonstrated that p210 BCR/ABL1 interacts directly with the xeroderma pigmentosum group B (XPB) protein, and that XPB is phosphorylated on tyrosine in cells that express p210 BCR/ABL1. In the current study, we have constructed a p210 BCR/ABL1 mutant that can no longer bind to XPB. The mutant has normal kinase activity and interacts with GRB2, but can no longer phosphorylate XPB. Loss of XPB binding is associated with reduced expression of c-MYC and reduced transforming potential in ex-vivo clonogenicity assays, but does not affect nucleotide excision repair in lymphoid or myeloid cells. When examined in a bone marrow transplantation (BMT) model for chronic myelogenous leukemia, mice that express the mutant exhibit attenuated myeloproliferation and lymphoproliferation when compared with mice that express unmodified p210 BCR/ABL1. Thus, the mutant-transplanted mice show predominantly neutrophilic expansion and altered progenitor expansion, and have significantly extended lifespans. This was confirmed in a BMT model for B-cell acute lymphoblastic leukemia, wherein the majority of the mutant-transplanted mice remain disease free. These results suggest that the interaction between p210 BCR/ABL1 and XPB can contribute to disease progression by influencing the lineage commitment of lymphoid and myeloid progenitors.