Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.

BeachWin: modelling groundwater effects on swash sediment transport and beach profile changes

Field and laboratory observations have shown that a relatively low beach groundwater table enhances beach accretion. These observations have led to the beach dewatering technique (artificially lowering the beach water table) for combating beach erosion. Here we present a process-based numerical model that simulates the interacting wave motion on the beach. coastal groundwater flow, swash sediment transport and beach profile changes. Results of model simulations demonstrate that the model replicates accretionary effects of a low beach water table on beach profile changes and has the potential to become a tool for assessing the effectiveness of beach dewatering systems. (C) 2002 Elsevier Science Ltd. All rights reserved.

CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappa B

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-kappaB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-alpha, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-kappaB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-kappaB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-alpha treatment, and this was NF-kappaB dependent. Whereas RelB/p50 translocation induced by TNF-alpha was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IkappaBalpha following CD40L stimulation. However, NF-kappaB activation induced by TNF-alpha could be sustained by blocking autocrine IL-10. These data indicate that NF-kappaB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-kappaB activation is prolonged. Because IL-10 moderates DC NF-kappaB activation by TNF-alpha, sustained NF-kappaB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-alpha.

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-systern in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-systern open to the outside, the sealed t-systern of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres. (C) 2002 Elsevier Science Ltd. All rights reserved.

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 muM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference,map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: Phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (K-i 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.

Is cell surface calreticulin involved in phagocytosis by insect hemocytes?

The innate immune system of insects consists of humoral and cellular components involved in the recognition of and responses to intruding foreign micro- or macroorganisms. Several molecules have been identified so far that recognize molecular patterns present on microorganisms, such as lipopolysaccharides, peptidoglycans and lipoteichonic acid. These molecules, acting as opsonins, trigger immune responses such as phagocytosis, nodule formation, melanization and encapsulation. Here, we investigated the role of calreticulin (CRT) present on the surface of Pieris rapae hemocytes in phagocytosis. Comparative phagocytosis assays using yeast cells showed that hemocytes from different insects exhibit significant variation in their phagocytosing potential and relative CRT involvement. (C) 2003 Elsevier Science Ltd. All rights reserved.

Association of increased levels of heavy-chain ferritin with increased CD4(+) CD25(+) regulatory T-Cell levels in patients with melanoma

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4+ CD25+ CD69- T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.