Electronic Devices Based on Purified Carbon Nanotubes Grown By High Pressure Decomposition of Carbon Monoxide

The excellent properties of transistors, wires, and sensors made from single-walled carbon nanotubes (SWNTs) make them promising candidates for use in advanced nanoelectronic systems. Gas-phase growth procedures such as the high pressure decomposition of carbon monoxide (HiPCO) method yield large quantities of small diameter semiconducting SWNTs, which are ideal for use in nanoelectronic circuits. As-grown HiPCO material, however, commonly contains a large fraction of carbonaceous impurities that degrade properties of SWNT devices. Here we demonstrate a purification, deposition, and fabrication process that yields devices consisting of metallic and semiconducting nanotubes with electronic characteristics vastly superior to those of circuits made from raw HiPCO. Source-drain current measurements on the circuits as a function of temperature and backgate voltage are used to quantify the energy gap of semiconducting nanotubes in a field effect transistor geometry. This work demonstrates significant progress towards the goal of producing complex integrated circuits from bulk-grown SWNT material.Comment: 6 pages, 4 figures, to appear in Nature Material

Purification and characterization of mast cell tryptase and chymase from human tissues

Mast cells are key effector cells of the allergic response. When stimulated by specific allergen through the high-affinity IgE receptors or through other stimuli, these cells release a number of potent mediators of inflammation. Amongst these are the serine proteases tryptase and chymase. In humans, tryptase is the most abundant mediator stored in mast cells. Chymase is present in more moderate amounts in a subpopulation of mast cells (MC(TC)). This subtype of mast cells predominates in connective tissue, whereas the other major subtype, the MC(T), predominates in mucosal tissue. Both proteases have been shown to act on specific extracellular proteins and peptides, as well as to alter the behavior of various cell types. Inhibitors of tryptase have been found to be efficacious in animal and human models of asthma, and both proteases are currently being investigated as potential targets for therapeutic intervention. Such pharmacological, physiological, and biochemical studies require the availability of purified tryptase and chymase.In this chapter, we shall describe procedures for the purification of tryptase and chymase from human tissues and provide protocols for monitoring purification and characterization of the final product. The preparation of recombinant proteases will not be covered, though some of the procedures described may be readily adapted for their purification from recombinant expression systems. The procedures described here have been developed for the purification of the human proteases and will require some modification if applied to purify mast cell proteases from the tissues of other species

Mass, charge, and subcellular localization of a unique secretory product identified by the basophil-specific antibody BB1

Background: BB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils. Objective: We sought to further characterize the basophilspecific antigen identified by BB1. Methods: Intracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing. Results: In flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 × 106 d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6. Conclusion: With its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein

Selective alterations in mast cell subsets and eosinophil infiltration in two complementary types of intestinal inflammation: ascariasis and Crohn's disease

Objective: Numbers of mast cells (MCs) of different subpopulations and the extent of eosinophil infiltration were compared in Crohn's disease and ascariasis. These two types of intestinal inflammation are complementary with regard to T cell response (TH1 versus TH2), prevalence and environmental factors.Methods: Histochemical, immunohistochemical and ultrastructural tools were applied to biopsies of morphologically uninvolved colon, ileum and duodenum from Crohn's and ascariasis patients, as well as resection margins and tissues from an experimental porcine ascariasis model. MC subsets were defined by their dye-binding properties, and their chymase content was analysed using biochemical tools.Results: The TH2 (IgE-mediated) response in ascariasis was characterised by a dramatic increase in mucosal- type MCs (MMCs) and eosinophils in both the mucosa and the deeper layers of the intestinal wall and a simultaneous decrease of connective tissue-type MCs (CTMCs). Uninvolved intestine of Crohn's patients showed moderate proliferation of CTMCs in the deeper layers of the intestinal wall, but a significant decrease of the MMCs, associated with moderate eosinophilia in all layers of the gut. Similar changes were present in the uninvolved duodenum of Crohn's patients. Comparable amounts of chymase could be extracted from mucosal and submucosal duodenum, with similar proportions of its two principal isoforms in each.Conclusions: Our results indicate that T cell responses (TH1 or TH2) are associated with different MC subsets in intestinal inflammation. Changes remote from the focus of inflammation point to the systemic nature of the different MC responses

Segmental bronchoprovocation in allergic rhinitis patients affects mast cell and basophil numbers in nasal and bronchial mucosa

Mast cells and basophils are cells that play an important role in the initiation and control of allergic inflammation in asthma and rhinitis. This study was undertaken to determine the presence and dynamics of mast cells and basophils in the nasal and bronchial mucosa of allergic rhinitis patients after segmental bronchial provocation (SBP). Eight nonasthmatic, grass pollen-allergic rhinitis patients and eight healthy controls were included. Bronchial and nasal biopsies, as well as blood samples, were taken before (T0) and 24 h (T24) after SBP. Immunohistochemical staining was performed for mast cells (tryptase and chymase; phenotypes MCT, MCTC, MCC) and basophils (BB1). In the bronchial mucosa, the number of BB1+ cells increased significantly (p < 0.05) in allergic rhinitis patients after SBP. In the nasal mucosa, the numbers of MCC and MCTC cells decreased significantly, whereas the numbers of [BB1+] cells increased significantly in allergic rhinitis patients after SBP (p < 0.05). In blood, the number of basophils decreased (p < 0.05) and the level of interleukin (IL)-5 increased (p < 0.05) in atopic patients after SBP. No significant changes could be observed in healthy controls. This study shows that SBP in nonasthmatic allergic rhinitis patients reduces numbers of mast cells in the nose as a result of enhanced degranulation. At the same time, there is evidence for an influx of basophils from the blood into the nasal and bronchial mucosae

Chemokine-induced cutaneous inflammatory cell infiltration in a model of Hu-PBMC-SCID mice grafted with human skin

Recently, certain chemokines and chemokine receptors have been preferentially associated with the selective recruitment in vitro of type 1 T cells, such as IP-10 and its receptor CXCR3, or type 2 T cells such as monocyte-derived chemokine (MDC) and eotaxin and their receptors CCR4 and CCR3. Very few models have provided confirmation of these findings in vivo. Taking advantage of the humanized SCID mouse model grafted with autologous human skin, the ability of the chemokines IP-10, MDC, eotaxin, and RANTES to stimulate cell recruitment was investigated. Intradermal IP-10 injection resulted in an influx of CD4+ T lymphocytes but also surprisingly in the recruitment of dendritic cells. MDC recruited mainly CD8+ T lymphocytes, and had little effect on eosinophils. As predicted, eotaxin was a potent inducer of eosinophil and basophil migration, also recruiting CD4+ T cells. RANTES, a ubiquitous chemokine associated with both type 1 and type 2 profiles, was able to recruit all cell types. CXCR3-positive cells were preferentially recruited by IP-10, whereas CCR3- and CCR4-positive cells were predominantly found after injection of eotaxin and MDC. Thus, in a human environment in vivo, some chemokines have the ability to recruit cells expressing chemokine receptors preferentially expressed on type 1 or type 2 cells. Further investigations revealed that MDC and eotaxin induced the recruitment of type 2, but not type 1, cytokine-producing cells. RANTES, on the other hand, induced the migration of both type 1 and type 2 cytokine-secreting cells, whereas IP-10 did not induce the recruitment of either subtype. These studies provide detailed information on the properties of MDC, eotaxin, IP-10, and RANTES as chemotactic molecules in skin in vivo. The use of the humanized SCID mouse model grafted with human skin is validated as a useful model for the evaluation of chemokine function in the inflammatory reaction, and suggests that therapeutic targeting of certain chemokines might be of interest in diseases associated preferentially with a type 1 or type 2 profile

Chemokine-Induced Cutaneous Inflammatory Cell Infiltration in a Model of Hu-PBMC-SCID Mice Grafted with Human Skin

Recently, certain chemokines and chemokine receptors have been preferentially associated with the selective recruitment in vitro of type 1 T cells, such as IP-10 and its receptor CXCR3, or type 2 T cells such as monocyte-derived chemokine (MDC) and eotaxin and their receptors CCR4 and CCR3. Very few models have provided confirmation of these findings in vivo. Taking advantage of the humanized SCID mouse model grafted with autologous human skin, the ability of the chemokines IP-10, MDC, eotaxin, and RANTES to stimulate cell recruitment was investigated. Intradermal IP-10 injection resulted in an influx of CD4(+) T lymphocytes but also surprisingly in the recruitment of dendritic cells. MDC recruited mainly CD8(+) T lymphocytes, and had little effect on eosinophils. As predicted, eotaxin was a potent inducer of eosinophil and basophil migration, also recruiting CD4(+) T cells. RANTES, a ubiquitous chemokine associated with both type 1 and type 2 profiles, was able to recruit all cell types. CXCR3-positive cells were preferentially recruited by IP-10, whereas CCR3- and CCR4-positive cells were predominantly found after injection of eotaxin and MDC. Thus, in a human environment in vivo, some chemokines have the ability to recruit cells expressing chemokine receptors preferentially expressed on type 1 or type 2 cells. Further investigations revealed that MDC and eotaxin induced the recruitment of type 2, but not type 1, cytokine-producing cells. RANTES, on the other hand, induced the migration of both type 1 and type 2 cytokine-secreting cells, whereas IP-10 did not induce the recruitment of either subtype. These studies provide detailed information on the properties of MDC, eotaxin, IP-10, and RANTES as chemotactic molecules in skin in vivo. The use of the humanized SCID mouse model grafted with human skin is validated as a useful model for the evaluation of chemokine function in the inflammatory reaction, and suggests that therapeutic targeting of certain chemokines might be of interest in diseases associated preferentially with a type 1 or type 2 profile

Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells

Airway remodeling with smooth muscle cell (SMC) hyperplasia is a feature of chronic asthma. We investigated the potential for tryptase, the major secretory product of human mast cells, to act as a growth factor for human airway SMCs. Because this serine protease can activate proteinase-activated receptor-2 (PAR-2), we also examined the actions of SLIGKV, a peptide agonist of PAR-2. Incubation with lung tryptase provoked a twofold increase in [3H]thymidine incorporation; a similar increase in cell numbers was found when we used the MTS assay. The effect was catalytic site dependent, being abolished by the protease inhibitors leupeptin and benzamidine and by heat inactivation of the enzyme. Tryptase-induced DNA synthesis was inhibited by preincubation of the cells with pertussis toxin, calphostin C, or genistein. Transduction mechanisms are thus likely to involve a pertussis toxin-sensitive G protein, protein kinase C, and tyrosine kinase. SLIGKV elicited a response on SMCs similar to that of tryptase. Tryptase could provide an important stimulus for SMC proliferation in asthmatic airways, by acting on PAR-2