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Notes: Summary We describe a 14-year-old boy who developed total leucoderma during the course of chronic graft versus- host disease, which developed after allogeneic bone marrow transplantation. Skin biopsy and dihydroxy-phenylalanine staining revealed a total absence of melanocyte from the epidermis. Cytotoxic anti-melanocyte antibodies were found in the patient's serum, and this probably explains the development of the leucoderma.

Notes: Background Mast cells are responsible for eliciting the early phase and for contributing to the development of the late phase of allergic reactions, through the release of cytokines and other inflammatory mediators.Objective To assess whether the glucocorticoid dexamethasone has a direct effect on mast cell progenitor maturation and on mature cord blood-derived mast cell properties.Methods Mast cells were obtained by culturing human umbilical cord blood mononuclear cells with stem cell factor, IL-6 and prostaglandin E2. Mast cell numbers were assessed by Toluidine Blue staining and immunocytochemistry of tryptase positive cells. The expression of FcεRI, CD49d and c-kit was assessed by flow cytometry. Histamine release was determined by a radioenzymatic assay. Cys-LT, GM-CSF and TNF-α production and release were determined by ELISA.Results Dexamethasone (10−6 M−10−9 M) time- and dose-dependently inhibited the maturation of the mast cell progenitors. Dexamethasone did not affect the basal expression of FcεRI, CD49d and c-kit, but it inhibited the IgE-dependent enhanced expression of FcεRI. Dexamethasone (10−6 M−10−9 M) had no significant effect on FcεRI-dependent histamine release or the synthesis and release of Cys-LT from the mature mast cells. However, pre-incubation of the mast cell cultures with dexamethasone for 1 h, prior to cross-linking of FcεRI, dose-dependently inhibited the production and secretion of both GM-CSF and TNF-α.Conclusions From these in vitro data we propose that glucocorticosteroids are effective drugs in the management of allergic inflammation due to their capacity to inhibit mast cell development, IgE-dependent FcεRI expression and mast cell production of GM-CSF and TNF-α.

Notes: Background Mast cells, the key cells of immediate hypersensitivity type reactions, have also been postulated to have a central role in influencing tissue remodelling and fibrosis occurring in the skin.Objective Our aim was to investigate the direct role of human mast cells (HMC) in skin fibrotic processes, by assessing the effects of the addition of the human mast cell line HMC-1 to human skin fibroblasts, and to identify the responsible mediators.Methods HMC-1 sonicates were added to human skin fibroblasts and the following parameters were evaluated: proliferation ([3H]-thymidine), collagen synthesis ([3H] proline), activity of matrix metalloproteinases (MMPs) (zymography) and tissue inhibitors of metalloproteinases (TIMPs) (reverse zymography), and collagen gel contraction.Results HMC-1 sonicate increased significantly both proliferation and collagen production in the human skin fibroblasts and these properties were not affected by heating of the sonicate (56 °C, 30 min, or 100 °C, 3 min). Two main mast cell mediators, histamine and tryptase, were found to be responsible for the increase in fibroblast proliferation and collagen production. HMC-1 sonicate did not display any pre-formed gelatinase activity, and its addition to the fibroblasts did not change their pro-MMP-2 and MMP-2 activity. On the other hand, HMC-1 were found to possess TIMP-1 and TIMP-2. Addition of HMC-1 had no effect on fibroblasts TIMP-1 but induced a dose-dependent increase of TIMP-2 activity. In addition, HMC-1 sonicate seeded together with the fibroblasts in tri-dimensional collagen gel significantly enhanced their contraction.Conclusion We have shown that human mast cells, by granule-stored and therefore quickly releasable mediators, increase human skin fibroblast proliferation, collagen synthesis, TIMP-2 and collagen gel contraction. Therefore, mast cells have a direct and potentiating role in skin remodelling and fibrosis.

Notes: Background In vivo rush desensitization is a procedure widely employed to quickly desensitizo allergic patients by administering increasing doses of the offending antigen at short intervals. The mechanism(s) underlying this process and the possible role of mast cells in it have not been well delineated.Objective To define an ex vivo model for rush desensitization utilizing the mast cell-fibroblast coculture system.Methods Rat peritoneal mast cells were cultured on 3T3 fibroblasls and were pre-incubated with saturating or suboptimal concentrations of IgE anti-DNP antibodies. Mast cells were then repeatedly challenged at 30 min intervals, with increasing amounts (0.001 100ng/mL) of the antigen DNP-HSA, in the presence of calcium ions. Parallel control cultures were stimulated only once by each antigen concentration. In another set of experiments, mast cells were repeatedly activated with increasing concentrations (0.1-1000 ng/mL) of compound 48/80. Supernatants and cell sonicates were assessed for histamine content and the percentage of histamine released was calculated.Results When saturating concentrations of IgE anti-DNP antibodies were used, mast cells challenged repeatedly with DNP-HSA did not release significant amounts of histamine up to an antigen concentration of I ng/mL. At this stage they were partially desensitized, releasing only 108.3 . 17.1 ng histamine/plate (7.9±0.8%). A marked desensitization was observed at optimal antigen concentration (100 ng/mL). where experimental mast cells released only 45.6±10.9ng/plate, compared with 661.9±48.2ng/plate in firstly challenged cultures. Desensitization was probably not due to mast cells histamine depletion, since the cells still contained large amounts of histamine (579.5±108.6ng/plate) at the end of the procedure. A similar pattern of desensitization was observed when mast cell were preincubated with a subotimal concentration of IgE antibodies. Activation of mast cells with increasing amounts of the IgE-independent secretagogue, compound 48/80. did not cause desensitization since at each concentration both repetitively challenged and control cultures released similar amounts of histamine. Furthermore, challenge of antigen-desensitized mast cells with compound 48/80 caused the release of 75.9±4.9% histamine comparable to the percentage of histamine released from controls (79.5±6.7).Conclusion Repeated exposure of mast cells to gradually increasing amounts of antigen induces their refractoriness. This observation would suggest a role for mast cells in rush desensitization procedure in vivo. Our coculture system may serve as a useful model for studying this process.

Notes: Background The amniotic membrane (AM), which is the innermost layer of the placenta, was shown to possess anti-inflammatory and anti-fibrotic properties in various in vitro and clinical studies.Purpose To evaluate the anti-fibrotic and anti-inflammatory effects of the AM matrix (AMM) on human conjunctival and lung fibroblasts in an in vitro system that tests fibrotic and inflammatory responses at the effector stages of allergic inflammation.Methods Human conjunctival or lung fibroblasts were seeded on plastic or on the stromal aspect of the AM, which was mounted on plastic inserts. Sonicates of human peripheral blood eosinophils activated with lipopolysacharide (LPS), or human mast cell (HMC-1) leukaemia cell sonicates, were added to sub-confluent fibroblast monolayers. Proliferation of the sub-confluent fibroblasts was assessed using the [3H]-thymidine incorporation assay. The production of transforming growth factor (TGF)-β1, granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-8 in conjunctival or lung fibroblasts was measured in conditioned media from these cultures by ELISA.Results After 4 days in culture, the [3H]-thymidine incorporation assay indicated a reduced proliferation of activated conjunctival and lung fibroblasts when cultured directly on the AMM. The production of both TGF-β1 and IL-8 was significantly suppressed in activated conjunctival fibroblasts cultured on the AMM compared with those cultured on plastic, while the production of both TGF-β1 and GM-CSF was decreased in human lung fibroblast cultured on the AMM.Conclusions The AMM is capable of suppressing fibrotic responses in an in vitro system of effector stages of ocular allergic inflammation. These data may provide a basis for exploring matrix components in the AM for the treatment of allergic eye disease.