Computing the centerline of a colon: a robust and efficient method based on 3D skeletons.

We present a robust and efficient algorithm for calculating the centerline of a computer-generated colon model created from helical CT image data. The centerline is an essential aid for navigating through complex anatomy such as the colon. Our algorithm involves three steps. In the first step, we generate a 3D skeleton of the binary colon volume using a fast topological thinning algorithm. In the second step, we employ a graph search algorithm to remove extra loops and branches. These loops and branches are caused by holes in the object that are artifacts produced during image segmentation. In the final step, we compute a smooth representation of the centerline by approximating the skeleton with cubic B-splines. This final step is necessary because the skeleton contains many abrupt changes in direction due to the discrete nature of image data. The user supplies two endpoints for the centerline; otherwise, the algorithm is fully automated. Experimental results demonstrate that the algorithm is not only robust but also efficient.

Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity.

This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.

Effects of cyclosporin A and dinactin on T-cell proliferation, interleukin-5 production, and murine pulmonary inflammation.

We compared the effects of cyclosporin A (CSA) and a macrotetrolide antibiotic, dinactin, on human T-cell proliferation and cytokine production induced by stimulation of the T-cell receptor alone (monoclonal antibody [mAb] directed against CD3) or in combination with costimulatory signals (mAbs directed against CD3 and CD28). These agents were also examined in a murine model of interleukin (IL)-5-mediated pulmonary inflammation. Dinactin inhibited T-cell proliferation induced by IL-2, by mAb to CD3, and by mAbs to CD3 plus alpha-CD28 with identical dose-response curves (IC50 = 10-20 ng/ml). Dinactin inhibited cytokine production with IC50 values of 10 ng/ml for IL-4 and IL-5 and 30 or 60 ng/ml for interferon-gamma or IL-2, respectively. Unlike CSA, exogenous IL-2 did not alter the dinactin-mediated effects on T cells, and nuclear run-on and steady-state messenger RNA (mRNA) analysis showed that dinactin inhibited cytokine production through a post-transcriptional mechanism. CSA selectively blocked T-cell receptor-induced T-cell proliferation and cytokine production (IC50 = 10 ng/ml). Under costimulatory conditions, IL-5 synthesis was only minimally inhibited by high concentrations of CSA, and at CSA concentrations of less than 125 ng/ml, IL-5 was significantly increased above control values. Dinactin and CSA reduced pulmonary eosinophilia when administered within 1 d of airway antigen challenge. Of the cytokine mRNAs examined in the lungs of CSA-pretreated, antigen-challenged mice, IL-5 mRNA levels were the least reduced, paralleling the resistance of IL-5 to CSA observed in vitro and suggesting a role for CD28 in the in vivo induction of IL-5.

Effects of chronic anti-interleukin-5 monoclonal antibody treatment in a murine model of pulmonary inflammation.

The maturation of eosinophils in bone marrow, their migration to pulmonary tissue, and their subsequent degranulation and release of toxic granule proteins contributes to the pathophysiology observed in asthma. Interleukin-5 (IL-5) is essential for these processes to occur. Therefore, much emphasis has been placed on attempts to inhibit the production or activity of IL-5 in order to attenuate the inflammatory aspect of asthma. In this report, the immunological consequences of long-term exposure to an antibody recognizing IL-5 (TRFK-5) were studied in a murine pulmonary inflammation model. A single dose of TRFK-5 (1 mg/ kg, intraperitoneally) reversibly inhibited antigen-dependent lung eosinophilia in mice for at least 12 wk and inhibited the release of eosinophils from bone marrow for at least 8 wk. Normal responses to aerosol challenge were attained after 24 wk. In mice treated acutely with antibody (2 h before challenge), 50% inhibition of pulmonary eosinophilia occurred when 0. 06 mg/kg TRFK-5 was administered (intraperitoneally; ED50), resulting in 230 ng/ml (IC50) in serum. In mice treated with one dose of TRFK-5 (1 mg/kg) and rested before challenge, the antibody exhibited a half-life of 2.4 wk. After 18 to 19 wk, antigen challenge-induced eosinophilia was inhibited by 50% and serum levels of TRFK-5 were 25 ng/ml. TRFK-5 remaining in mice 8 wk after a single injection of TRFK-5 was sufficient to inhibit at least 50% of the eosinophilia induced in blood 3 h after injection of recombinant murine IL-5 (10 microg/kg, intravenously). To assess the biologic effect of long-term exposure of mice to antibody, several parameters of immune-cell function were measured. Throughout the extended period of activity of TRFK-5 (>/= 12 wk) there were no gross effects on antigen-dependent increases in T-cell recruitment into bronchoalveolar fluid (BALF), in IL-4 and IL-5 steady-state mRNA levels in lung tissue, or in immunoglobulin E (IgE) and IgG levels in serum. There was a small increase in IL-5 steady-state mRNA production in TRFK-5-treated mice after 2 h or 2 wk, but this was not observed at other times examined. In untreated mice, IL-5 steady-state mRNA production in response to antigen challenge decreased > 6-fold with age, although at all time points there was an increase in mRNA levels following challenge. Therefore, at later times, 25 ng/ml rather than 230 ng/ml of TRFK-5 inhibited BALF eosinophilia, probably because of reduced IL-5 levels. Twenty-four weeks after treatment with TRFK-5, when challenge-induced eosinophilia was restored, there was an excess of CD4(+) T cells in BALF from challenged mice. However, these T cells had no measurable effects on other responses to challenge, including cytokine production, B-cell accumulation, and immunoglobulin production in serum. Thus, the biologic duration of TRFK-5 was several months, and its activity was due to the presence of antibody above a therapeutic threshold rather than to any profound effect on the immune system.

Anti-inflammatory activity of inhaled mometasone furoate in allergic mice.

Mometasone furoate (CAS 83919-23-7, Sch 32088) is a new inhaled corticosteroid that is being developed to treat allergic inflammatory airway disorders such as rhinitis and asthma. In this study, we investigated the effects of inhaled mometasone furoate in allergic mice that, after antigen challenge, develop an influx of eosinophils and T cells and display an increased mRNA expression of proinflammatory cytokines in the lungs. Mometasone furoate aerosol was generated from metered dose inhalers and delivered into an animal exposure chamber. The mice were exposed to mometasone furoate by nose-only inhalation at respired doses ranging from 0.5-33 micrograms/kg given 24, 18 and 2 h before aeroallergen challenge. The elevated eosinophil numbers in the bronchoalveolar lavage fluid and lung tissues of sensitized, ovalbumin challenged mice were dose-dependently inhibited by inhaled mometasone furoate. Increased numbers of Thy1+ T cells and CD4+ (T-helper) and CD8+ (T-cytotoxic) T cell subsets were seen in the bronchoalveolar lavage fluid of ovalbumin-challenged mice. Pretreatment of these animals with mometasone furoate (33 micrograms/kg) reduced the number of Thy1+ T cells and the T-helper subset. Furthermore, mometasone furoate (33 micrograms/kg) reduced the percentage of CD44+ T-helper cells (activated/memory cells) to the levels observed in non-sensitized, ovalbumin-challenged mice. There were increased levels of steady-state mRNA for interleukin-4, interleukin-5, and to a lesser extent, gamma-interferon in the lungs of sensitized mice after ovalbumin challenge and pretreatment with mometasone furoate reduced the steady-state mRNA levels of these cytokines. Our results demonstrate a potent lung anti-inflammatory effect of inhaled mometasone furoate and identify that inhibition of T cell influx, eosinophil accumulation and modulation of cytokine activity are important components of this response.

Eosinophils retain their granule major basic protein in a murine model of allergic pulmonary inflammation.

Accumulation of eosinophils in the lung with concomitant tissue damage are defining histopathologic features of human asthma. Through degranulation and the release of proinflammatory proteins such as major basic protein (MBP), eosinophils may perpetuate this inflammatory response. We investigated the extent of eosinophil degranulation in a murine model of allergic pulmonary inflammation. In this paradigm, the mice develop pulmonary eosinophilia, mucus hypersecretion, tissue damage, and airway edema and hyperreactivity. To evaluate the degree of eosinophil degranulation, we used a polyclonal antibody to murine MBP (mMBP) to perform dot blot analysis of bronchoalveolar lavage (BAL) cells and fluids, and immunohistochemical fluorescent analysis of lung tissue sections. After ovalbumin antigen challenge, we were unable to detect immunoreactive mMBP in the BAL fluids from either nonsensitized or sensitized mice. However, after lysis of the recoverable BAL cells, we were able to detect mMBP by immunoblot analysis, with the levels of immunoreactive mMBP directly related to the number of recoverable eosinophils. We also examined paraffin-embedded, lung tissue sections for patterns of mMBP deposition. Whereas lung sections from allergic mice revealed prominent peribronchial eosinophilia after antigen challenge, tissue sections from nonsensitized animals rarely displayed eosinophils. Despite the presence of numerous eosinophils, no immunohistologic evidence of extracellular mMBP could be found in antigen-challenged allergic mice. Furthermore, rechallenged allergic mice displayed a significant increase in the number of recruited pulmonary eosinophils but all immunoreactive mMBP was still intracellular. We conclude that the recruited pulmonary eosinophils have not substantially degranulated. These results suggest that, in this murine model of allergic inflammation, eosinophil degranulation and release of mMBP does not contribute to the observed pulmonary inflammation and airway hyperreactivity.

Role of nitric oxide on eosinophilic lung inflammation in allergic mice.

Nitric oxide (NO) is an important mediator of inflammatory reactions and may contribute to the lung inflammation in allergic pulmonary diseases. To assess the role of NO in pulmonary inflammation, we studied the effect of four nitric oxide synthase (NOS) inhibitors, N-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, N(G)-monomethyl-L-arginine (NMMA) and L-N6-(1-Iminoethyl) lysine (L-NIL), on the influx of eosinophils into the bronchoalveolar lavage (BAL) fluid and lung tissue of antigen-challenged allergic mice. We also analyzed lung tissues for the presence of steady state mRNA for inducible nitric oxide synthase (iNOS) and iNOS protein. Furthermore, BAL fluid and serum were analyzed for their nitrite content. B6D2F1/J mice were sensitized to ovalbumin (OVA) and challenged with aerosolized OVA. The NOS inhibitors were given 0.5 h before and 4 h after the antigen challenge. OVA challenge induced a marked eosinophilia in the BAL fluid and lung tissue 24 h after challenge. The OVA-induced pulmonary eosinophilia was significantly reduced by L-NAME (10 and 50 mg/kg, intraperitoneally [i.p.]). The inactive isomer, D-NAME (50 mg/kg, i.p.) had no effect. When mice were treated with L-NAME (20 mg/kg, i.p.) and an excess of NOS substrate, L-arginine (200 mg/kg, i.p.), the OVA-induced pulmonary eosinophilia was restored. Treatment with aminoguanidine (0.4-50 mg/kg, i.p.) also reduced the pulmonary eosinophilia. Treatment with NMMA (2-50 mg/kg, i.p.) partially reduced the eosinophilia, but L-NIL (10-50 mg/kg, i.p.), a selective iNOS inhibitor, had no effect. L-NAME had no effect on the reduction of eosinophils in the bone marrow following OVA challenge to sensitized mice. OVA challenge to sensitized mice had no effect on iNOS protein expression or iNOS mRNA in the lungs or on the levels of nitrite in the BAL fluid. These results suggest that NO is involved in the development of pulmonary eosinophilia in allergic mice. The NO contributing to the eosinophilia is not generated through the activity of iNOS nor does NO contribute to the efflux of eosinophils from the bone marrow in response to antigen challenge. It is speculated that after antigen challenge, the localized production of NO, possibly from pulmonary vascular endothelial cells, is involved in the extravasation of eosinophils from the circulation into the lung tissue.

Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody.

Interleukin-5 (IL-5) is important in the control of differentiation, migration, and activation of eosinophils. In order to study the role of IL-5 in the development of eosinophilic inflammation of the airways, we have used a monoclonal antibody to murine IL-5 (TRFK-5) in a murine model of allergic pulmonary inflammation. B6D2F1 mice were sensitized with alum-precipitated ovalbumin and were challenged with aerosolized ovalbumin on day 12 after sensitization. Samples of bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow aspirate were collected at different times after ovalbumin challenge. Twenty-four hours after challenge there were significant increases in the number of eosinophils in the BAL fluid, lung tissue, and blood while bone marrow eosinophils were decreased. Treatment of sensitized mice with TRFK-5 (0.01-1 mg/kg, i.p.) 2 h before ovalbumin challenge reduced the numbers of eosinophils in the BAL fluid and lung tissue and prevented the decrease in bone marrow eosinophils in a dose-dependent fashion. The number of eosinophils in the BAL fluid, peribronchial and alveolar regions of the lung was also reduced when TRFK-5 (2 mg/kg, i.p.) was given up to 5 d after ovalbumin challenge. Furthermore, there was no evidence of increased epithelial damage, edema, or the presence of mucus that could have resulted from eosinophil apoptosis and release of toxic proteins after neutralization of IL-5. These results demonstrate an important role for IL-5 in the development of eosinophilic inflammation of the airways and for the migration of eosinophils from the bone marrow into blood in response to antigen challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cells modulate allergic pulmonary eosinophilia in mice.

Mast cells are important effector cells in IgE-mediated acute allergic reactions. Mast cells also produce cytokines such as interleukin (IL)-3, IL-4, IL-5, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) that regulate the function of eosinophils and the development of a late-phase inflammatory response to antigen challenge. To evaluate the role of mast cells on the development of IgE-mediated allergic pulmonary eosinophilia in vivo, we compared the eosinophil infiltration into lungs of mast cell deficient mice (WBB6F1/J-W/Wv) with their congenic normal littermates (W/W+). Mice were sensitized with alum-precipitated ovalbumin and challenged with aerosolized ovalbumin on day 12 after sensitization. Bronchoalveolar lavage (BAL) fluid, lung tissue biopsies, and blood samples were collected after ovalbumin challenge. Eosinophil numbers in the BAL and lung tissue, lung eosinophil peroxidase (EPO) activity and serum levels of IgE and IgG1 were measured. In sensitized W/W+ mice, there were increased numbers of eosinophils in the BAL fluid and lung tissue, and EPO levels were increased after ovalbumin challenge. Ovalbumin challenge of sensitized mast-cell-deficient mice produced fewer numbers of eosinophils in the BAL fluid and lungs, and EPO levels were also reduced compared with their challenged congenic littermates. On the other hand, levels of serum IgE and IgG1 were not different between W/Wv mice and their congenic littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

T cells are necessary for Th2 cytokine production and eosinophil accumulation in airways of antigen-challenged allergic mice.

In a murine model of pulmonary inflammation, aerosolized antigen challenge of sensitized B6D2F1 mice leads to eosinophil accumulation within the lungs. Little is known of the role of T cells and their cytokine products in these allergic animals. In this study, we show that T cells migrate into the lungs in response to antigen challenge and are necessary for local production of cytokines (IL-4 and IL-5) important in B and T cell development as well as eosinophil activation and differentiation. Flow cytometry revealed an increase in the percentage of Thy1+ T cells but not in B220+ B cells in bronchoalveolar lavage fluid after challenge when compared to unchallenged mice. Although there was an increase in both T cell subsets, there were twice as many CD4+ cells as CD8+ cells at 24 hr and after 48 hr the CD4+ subset predominated. The CD4+ T lymphocytes were CD44+ CD45RBlo indicating an activated/memory phenotype and tracheobroncheal lymph node cells obtained from challenged mice proliferated in a dose-dependent manner in response to antigen stimulation in vitro. Reverse transcriptase-polymerase chain reaction analysis of lung tissue-derived RNA indicated an increase in Th2-like cytokines. IL-4 and IL-5 steady-state mRNAs were at peak levels 6 hr after challenge, while no consistent increase was found for IFN-gamma mRNA levels. Treatment with the glucocorticoid betamethasone just prior to challenge reduced the levels of cytokine mRNA as well as the eosinophil influx. In vivo depletion of T cells from sensitized mice reduced pulmonary eosinophilia as well as the expression of IL-4, IL-5, and IFN-gamma steady-state mRNAs in the lungs of sensitized and challenged mice. These results indicate that T cells migrating into the lungs of mice after antigen challenge play an important role in the production of Th2-like cytokines and the accumulation of eosinophils in bronchial fluids.