Naoya Sakaguchi

Characterization of cord-blood-derived human mast cells cultured in the presence of Steel factor and interleukin-6.

We generated > 10(7) mast cells by culturing 10(7) cord blood mononuclear cells for > 10 weeks in the presence of Steel factor, interleukin-6 and prostaglandin E2. 99% of the cultured cells had tryptase-positive granules, while 18% had chymase-positive granules. Cultured mast cells contained 3.6 micrograms histamine and 3.5 micrograms tryptase per 10(6) cells. Cells sensitized with 1 microgram/ml human IgE released 58.5% histamine and 1.55 ng tumor necrosis factor (TNF)-alpha per 10(6) cells when challenged with 1 microgram/ml antihuman IgE, whereas the control cells spontaneously released 3.7% histamine and 0.18 ng TNF-alpha. Analysis for surface antigens revealed that cultured mast cells expressed the following CD molecules: 9, 13, 14, 29, 33, 38, 43, 44, 45RA, 45RB, 46, 47, 48, 49d, 50, 51, 53, 54, 55, 58, 59, 60, 61 and 117 (c-Kit). Taken together, these cultured cells seem to be functionally mature mast cells.

Extracellular ATP stimulates interleukin-dependent cultured mast cells and eosinophils through calcium mobilization.

We examined the effect of ATP and related nucleotides on the changes in intracellular calcium ([Ca2+]i) in murine bone marrow-derived mast cells (BMMC) and human cord blood-derived eosinophils (EO) cultured in the presence of interleukins. ATP, ADP and AMP released a substantial amount of histamine and leukotriene C4 from BMMC, and EO showed locomotive activity in response to ATP, ADP and GTP. These reactions were accompanied with an increase in [Ca2+]i in BMMC and in EO. The rise in [Ca2+]i in BMMC induced by ATP or antigen at optimal concentrations was inclined to be persisting. On the other hand, these nucleotides induced a rapid and transient rise in [Ca2+]i in EO. Purified human peripheral EO also exhibited locomotive activity and an increase in [Ca2+]i in response to ATP. These results indicate that extracellular ATP activates interleukin-dependent cultured mast cells and EO through Ca2+ mobilization, and suggest that ATP, which is known to be released from activated platelets or autonomic nerves, may stimulate in vivo counterparts of these cultured inflammatory cells.

Incidence of Latex Allergy in Atopic Children and Hospital Workers in Japan

The incidence of latex-induced allergy has been reported to be increasing in Europe and the US but not in Japan. We thus measured latex-specific IgE antibodies and latex-specific IgG antibodies in sera from 196 atopic children with low serum IgE levels (under 1,000 U/ml; group 1), 108 atopic children with high serum IgE levels (over 1,000 U/ml; group 2) and 601 hospital employees (group 3). Atopic children were diagnosed as having asthma, atopic dermatitis and/or food intolerance. One out of group 1 (0.5%) and 11 out of group 2 (10.2%) were found to have latex-specific IgE by radioallergosorbent assay (RAST), and 7 were further found to be positive for latex antigen by skin test. Fifty-five percent of group 1, 65% of group 2 and 9.7% of group 3 were found to have latex-specific IgG over 100 units/ml by enzyme linked immunosorbent assay (ELISA). Prior to our diagnosis most of the children and employees did not realize they were allergic to latex. These data suggest that caution should be taken regarding latex allergy when atopic children have to be operated upon as atopic children tend to be sensitized to the latex antigen after even minimal contact with latex products.

Continuous Isoproterenol Inhalation Therapy in Children with Severe Asthmatic Attack

We studied the 1-type isoproterenol inhalation therapy for patients with severe asthmatic attacks who were admitted at the Department of Allergy of National Childrens Hospital from 1981 to 1991. One hour after l-type isoproterenol inhalation therapy, statistically significant effects were noted with regard to the asthmatic status. Moreover, no side effect was found amoung the subjects. From these data, 1-type isoproterenol inhalation therapy is thought to be effective for severe asthmatic attacks.

Serum theophylline concentration levels and preventative effects on exercise‐induced asthma

One of the specific characteristics of asthma is bronchia! hyperresponsiveness. Many factors affect asthmatic patients [1]. Exercise is a very important triggering factor for bronchoconstriction in children. School-age asthmatic children are usually required to participate in various sports programmes [2]. There is much in the literature about exercise-induced asthma (EIA). and many studies have been published about the preventive effects and mechanisms of various drugs [3,4]. Many reports exist about exercise-induced early responses, but there is also a iate response phase [5]. The concept of late responses is not well accepted at present. However, in spite of careful treatment, some asthmatic patients may develop an exercise-induced late asthmatic response. The authors have already studied what drugs can be used to prevent exercise-induced late response, but there are no reports concerning bronchodilator effectiveness for preventing it. The authors investigated sustained-release theophylline effects on exercise-induced early and late responses. The effectiveness of the newly-proposed scrum theophylline therapeutic level of 5-15ng/ml, instead of 10-20|ig/ ml. was also studied by testing to sec whether a slightly greater than 5|.ig/nii serum thcophyllitie concentration would be effective at preventing EIA.

Growth in Methylcellulose of Human Mast Cells in Hematopoietic Colonies Stimulated by Steel Factor, a c-kit Ligand

We examined the effect of Steel factor (SLF) on the development of human mast cells in hematopoietic colonies from cord blood mononuclear cells in methylcellulose culture. When cord blood cells were cultured for 3 weeks, SLF increased the cellular tryptase levels detected in total cultured cells. It also stimulated the formation of small-cell colonies consisting mainly of polymorphonuclear granulocytes and immature blastoid cells in a concentration-dependent manner but not the formation of colonies consisting of large macrophagic cells. A low percentage of tryptase-positive mast-cell-like cells was found in 39 out of 100 granulocyte/blastoid cell colonies. Four of the 100 colonies contained 10-20% tryptase-positive cells, but we failed to observe colonies consisting of > 20% of tryptase-positive cells. These results suggest that the effect of SLF on mast cell growth is brought on by stimulating the growth of primitive hematopoietic progenitors.