Stochastic simulation of hemagglutinin-mediated fusion pore formation.

Studies on fusion between cell pairs have provided evidence that opening and subsequent dilation of a fusion pore are stochastic events. Therefore, adequate modeling of fusion pore formation requires a stochastic approach. Here we present stochastic simulations of hemagglutinin (HA)-mediated fusion pore formation between HA-expressing cells and erythrocytes based on numerical solutions of a master equation. The following elementary processes are taken into account: 1) lateral diffusion of HA-trimers and receptors, 2) aggregation of HA-trimers to immobilized clusters, 3) reversible formation of HA-receptor contacts, and 4) irreversible conversion of HA-receptor contacts into stable links between HA and the target membrane. The contact sites between fusing cells are modeled as superimposed square lattices. The model simulates well the statistical distribution of time delays measured for the various intermediates of fusion pore formation between cell-cell fusion complexes. In particular, these are the formation of small ion-permissive and subsequent lipid-permissive fusion pores detected experimentally (R. Blumenthal, D. P. Sarkar, S. Durell, D. E. Howard, and S. J., J. Cell Biol. 135:63-71). Moreover, by averaging the simulated individual stochastic time courses across a larger population of cell-cell-complexes the model also provides a reasonable description of kinetic measurements on lipid mixing in cell suspensions (T. Danieli, S. L. Pelletier, Y.I. Henis, and J. M. White, 1996, J. Cell Biol. 133:559-569).

Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease.

Previous studies on the frequency of intestinal mast cells and eosinophils in patients with inflammatory bowel disease yielded conflicting results. In the present morphometric study, we quantified mast cells and eosinophils in the lamina propria by histological and immunohistochemical methods in 64 patients suffering from Crohn's disease (33 cases) or ulcerative colitis (31 cases), and in 29 controls. Histological data from 206 biopsies were related to the presence of mucosal inflammation and clinical parameters. The number of eosinophils was increased in patients with inflammatory bowel conditions (mean +/- SE: 331 +/- 44/mm2) as compared to controls (258 +/- 27/mm2), and was dependent on disease activity and drug treatment. Mean mast cell numbers did not differ between patients and controls. However, a reduced mast cell number was found in toluidine blue-stained sections of actively inflamed tissue areas (143 +/- 16/mm2, versus 206 +/- 18/mm2 in non-inflamed tissue). Immunohistochemical studies using antibodies against the granule proteins tryptase and chymase suggest that this decrease in mast cell numbers is due to mast cell degranulation. The present data show that the number of intestinal mast cells and eosinophils is altered in patients with inflammatory bowel diseases, suggesting that both cell types are involved in the pathogenesis of chronic intestinal inflammation.

Assessment of fusogenic properties of influenza virus hemagglutinin deacylated by site-directed mutagenesis and hydroxylamine treatment.

Influenza virus hemagglutinin (HA) subtype H7 expressed from a baculovirus vector in insect cells requires cysteine residues for palmitoylation. Mutant HA devoid of fatty acids shows hemagglutinating and hemolytic activities almost identical to those of the acylated wild-type HA (wt). Using a membrane mixing assay (R18), neither the kinetics nor the pH dependence of fusion induced by wt or mutant HA was significantly different from virus-induced fusion. HA-induced fusion of insect cells with human erythrocyte ghosts could also be demonstrated by a cytoplasmic content mixing assay. Both species of recombinant HA induced the flow of lucifer yellow from preloaded ghosts into the cytoplasm of HA-bearing cells. This indicates that membrane fusion mediated by wild-type and fatty-acid-free HA includes both leaflets of the lipid bilayers. Hydroxylamine treatment of wt HA (H7) and fatty-acid-free mutant HA present in lysates of insect cells led to the complete inhibition of hemolytic activity. Deacylation of spike proteins by NH2OH treatment of virus particles resulted in a block of hemolytic activity in influenza virus subtypes H7 and H10 as well as of that in the togaviruses Semliki Forest and Sindbis virus. However, the same treatment did not affect subtypes H2 and H3 or two vesicular stomatitis virus serotypes. With such a differential effect whether or not fatty acids are present in the spike proteins of the different virus particles, hydroxylamine must have other effects than just deacylation, and therefore seems unsuitable for the study of the biological functions of acylproteins.