Design of potent inhibitors of human beta-secretase. Part 2.

We describe an optimized series of acyclic hydroxyethylamine transition state isosteres of beta-secretase that incorporates a variety of P(2) side chains that yield potent inhibitors with excellent cellular activity. A 2.2A crystal structure of compound 13 is shown.

Design of potent inhibitors of human beta-secretase. Part 1.

We describe a novel series of potent inhibitors of human beta-secretase. These compounds possess the hydroxyethyl amine transition state isostere. A 2.

Flow cytometric detection of antigen-specific cytokine responses in lung T cells in a murine model of pulmonary inflammation.

Multiparameter flow cytometry was used to examine the cytokine responses of antigen-specific T lymphocytes isolated from the lungs of antigen-sensitized mice which developed pulmonary inflammation after aerosol challenge with ovalbumin (OA) (OA/OA). Lung T cells were stimulated in vitro with OA and anti-CD28 monoclonal antibody (mAb) in the presence of the secretion inhibitor, brefeldin A. T cell subsets were examined for intracellular cytokine expression using fluorochrome-labeled cell-surface specific and anti-cytokine antibodies.

Preclinical evaluation of anti-inflammatory activities of the novel pyrrolopyrimidine PNU-142731A, a potential treatment for asthma.

The anti-inflammatory properties of a novel pyrrolopyrimidine, PNU-142731A, in a murine model of antigen-induced eosinophilic lung inflammation are described. PNU-142731A, when given orally, demonstrated a dose-related inhibition of eosinophil- and lymphocyte-rich accumulation in the airways of ovalbumin (OA)-sensitized and challenged (OA/OA) C57BL/6 mice. The magnitude of the suppression of lung inflammation was also dependent on length of treatment.

Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice.

Intercellular adhesion molecule-1-deficient mice have antibody responses but impaired leukocyte recruitment.

The role of intercellular adhesion molecule-1 (ICAM-1) in murine lung inflammation was examined in vivo. Ovalbumin (Ova)-sensitized and -challenged ICAM-1-deficient (KO) mice had decreased accumulation of leukocytes in the bronchoalveolar lavage fluid compared with wild-type (WT) mice. Lung tissue inflammation was also attenuated.

Identification of cytokine and adhesion molecule mRNA in murine lung tissue and isolated T cells and eosinophils by semi-quantitative reverse transcriptase-polymerase chain reaction.

We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect the expression of mRNA for inflammatory cytokines, integrins and selectins in murine lung tissue, and T cells and eosinophils isolated from lung and bronchoalveolar lavage (BAL) fluid in an in vivo model of ovalbumin (OA)-induced airway inflammation. RNA was isolated from whole lung tissue at 1, 6, 24, 48, 72 h, and 7 days after OA inhalation. mRNA for the Th2 cytokines, IL-4, -5, -6, -10 and -13 in OA-sensitized mice were significantly elevated compared with non-sensitized mice.

Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1.

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation.

Role of intercellular adhesion molecule-1 in antigen-induced lung inflammation in brown Norway rats.

We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype.

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells.

Phenotypic analysis of airway eosinophils and lymphocytes in a Th-2-driven murine model of pulmonary inflammation.

In order to investigate whether the pulmonary response to helminth antigens mimics that seen in allergic inflammation of the airways, we have examined the phenotypic characteristics of lymphocytes and eosinophils recruited to the airways following Nippostrongylus brasiliensis (N.b.) infection.

Phenotypic characterization of T lymphocytes emigrating into lung tissue and the airway lumen after antigen inhalation in sensitized mice.

Cytokines released from CD4+ T lymphocytes contribute to the pathogenesis of asthma by influencing the differentiation and function of eosinophils, the primary effector cells that cause airway epithelial damage. Using a model of ovalbumin (OA)-induced, eosinophil-rich chronic lung inflammation in sensitized mice, we have defined the role of T lymphocytes further by using three-color flow cytometry to characterize the adhesion and activation antigens that may be associated with the migration of these cells into the lung and airway lumen. OA inhalation in OA-sensitized C57BL/6 mice resulted in an early (6 to 24 h) influx of neutrophils into the bronchial lumen as enumerated by bronchoalveolar lavage (BAL), which was followed by a marked accumulation of lymphocytes and eosinophils between 24 to 72 h.

Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts.

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA.

T lymphocyte adhesion to human synovial fibroblasts. Role of cytokines and the interaction between intercellular adhesion molecule 1 and CD11a/CD18.

We studied the adhesion of human peripheral blood T lymphocytes to human synovial fibroblasts stimulated with interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or combinations of these cytokines. T lymphocytes bound poorly to untreated human synovial fibroblasts. IFN gamma treatment resulted in the largest increase in adhesion, followed by TNF alpha and IL-1 beta.

Beta-carotene's effects on serum lipoproteins and immunologic indices in humans.

Doses of beta-carotene for cancer-prevention trials have been chosen based on epidemiologic data. Mechanisms of the putative antineoplastic effects by beta-carotene are unknown but may involve modulation of the immune system. We measured plasma carotenoid concentrations and selected immunologic indices at baseline and at 2 and 4 wk in 50 healthy humans (5 groups of 10 each) ingesting 0, 15, 45, 180, or 300 mg beta-carotene/d for 1 mo in this randomized placebo-controlled, open-label, parallel study.

Role of cytokines in inflammatory synovitis. The coordinate regulation of intercellular adhesion molecule 1 and HLA class I and class II antigens in rheumatoid synovial fibroblasts.

This study was undertaken in an effort to understand the role of cytokines in T lymphocyte trafficking into inflamed synovium and in the potential enhancement of antigen presentation by human synovial fibroblasts. We found that interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN gamma) each increased the cell surface expression of intercellular adhesion molecule 1 (ICAM-1) on human synovial fibroblasts in a dose- and time-dependent manner. Maximal ICAM-1 expression occurred within 8 hours of induction, with the following order of efficacy: IFN gamma greater than TNF alpha greater than IL-1 beta.

In vivo administration of interleukin 1 elicits increased Ia antigen expression on B cells through the induction of interleukin 4.

The effects of the in vivo administration of interleukin 1 (IL 1) on lymphocytes from lymph node and spleen were analyzed. Mice received five daily subcutaneous (s.c.

Candida albicans-induced agglutinin and immunoglobulin E responses in mice.

Mice varied in their ability to make detectable antibody responses to cell surface determinants of Candida albicans depending upon the antigen preparation and the immunization schedule used. Immunoglobulin M (IgM) appeared to be the major class of antibody responsible for the C. albicans-agglutinating activity of the immune sera.