Airway expression of calcitonin gene-related peptide in T-cell peptide-induced late asthmatic reactions in atopics.

BACKGROUND: The mechanisms of late asthmatic reactions provoked in atopic asthmatics by allergen-derived T-cell peptide epitopes remain unclear. Previous studies showed no changes in airway eosinophils or mast cell products after peptide challenge. In the present study our aim was to measure calcitonin gene-related peptide (CGRP), neurokinin (NK)-A, and substance P (SP) in bronchoalveolar lavage fluid and bronchial biopsies (BB) after inhalation of allergen-derived T-cell peptide epitopes since these neuropeptides (NP) had not previously been evaluated in this chronic asthma model. METHODS: Bronchoscopy, with BB and bronchoalveolar lavage (BAL), was performed in 24 cat-allergic subjects 6 h after inhalation of Fel d 1-derived peptides. Neuropeptides were measured in BAL by enzyme-linked immunosorbent assay and CGRP expression in the airways was assessed by immunohistochemistry and confocal microscopy. RESULTS: Twelve subjects (termed 'responders') developed isolated late reactions. Calcitonin gene-related peptide, but not NK-A or SP, was significantly elevated in BAL in responders only. Biopsy studies showed that in virtually all responders peptide challenge induced marked increases in CGRP immunoreactivity in bronchial epithelial cells, infiltrating submucosal cells and in association with airway smooth muscle. Double immunostaining indicated that CGRP colocalized predominantly to CD3+/CD4+ and CD68+ submucosal inflammatory cells. CONCLUSION: Calcitonin gene-related peptide, a potent vasodilator, is markedly up-regulated in the airways of atopic asthmatics during late-phase reactions provoked by inhalation of allergen-derived T-cell peptides.

Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein.

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the coxsackievirus and Ad receptor (CAR), promotes virus attachment to cells and is a major determinant of Ad tropism. Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR together (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independently by surface plasmon resonance demonstrated that the IgV domain is necessary and sufficient for binding, and no additional membrane components are required to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber knob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG loop, and Leu485Lys in beta strand F, effectively abolished high-affinity binding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in beta strand E significantly reduced the level of binding. Circular dichroism spectroscopy showed that these mutations do not disorder the secondary structure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or indirectly involved in CAR binding. The critical residues have exposed side chains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or toward the edge of the R sheet. These findings have implications for our understanding of the biology of Ad infection, the development of novel Ad vectors for targeted gene therapy, and the construction of peptide inhibitors of Ad infection.

Mutations in the DG loop of adenovirus type 5 fiber knob protein abolish high-affinity binding to its cellular receptor CAR.

The amino acid residues in adenovirus type 5 (Ad5) fiber that interact with its cellular receptor, the coxsackie B virus and Ad receptor (CAR), have not been defined. To investigate this, multiple mutations were constructed in the region between residues 479 and 497 in Ad5 fiber (beta-strands E and F and the adjacent region of the DG loop). The effects of these mutations on binding to CAR were determined by use of cell-binding competition experiments, surface plasmon resonance, and direct binding studies. The mutation effects on the overall folding and secondary structure of the protein were assessed by circular dichroism (CD) spectroscopy. Deletions of two consecutive amino acids between residues 485 and 493 abolished high-affinity binding to CAR; the CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conformational changes did occur. Moreover, single site mutations in this region of residues with exposed, surface-accessible side chains, such as Thr492, Asn493, and Val495, had no effect on receptor binding, which demonstrates that these residues are not in contact with CAR themselves. This implies the involvement of residues in neighboring loop regions. Replacement of the segment containing the two very short beta-strands E and F and the turn between them (residues 479 to 486) with the corresponding sequence from Ad3 (betaEFAd3-->5 mutation) resulted in the loss of receptor binding. The identical CD spectra for betaEFAd3-->5 and wild-type proteins suggest that these substitutions caused no conformational rearrangement and that the loss of binding may thus be due to the substitution of one or more critical contact residues. These findings have implications for our understanding of the interaction of Ad5 fiber with CAR and for the construction of targeted recombinant Ad5 vectors for gene therapy purposes.

Cultured human airway smooth muscle cells stimulated by interleukin-1beta enhance eosinophil survival.

Airway smooth muscle may be an important cellular source of proinflammatory mediators and cytokines and may participate directly in airway inflammation. In this study we have examined whether airway smooth muscle cells could contribute to mechanisms of eosinophil accumulation by prolonging their survival. To investigate this possibility, conditioned medium from human airway smooth muscle cells stimulated with interleukin (IL)-1beta was examined on the in vitro survival of highly purified human peripheral blood eosinophils. After 7 d, when cultured in control medium, less than 1 +/- 0.2% of the initial eosinophil population remained viable. In contrast, culture in medium conditioned for 96 h by human airway smooth muscle cells stimulated with IL-1beta (1 pg-100 ng/ml) resulted in a concentration-dependent increase in eosinophil survival. (The concentration that produced 50% of this effect was 0.03 ng/ml IL-1beta.) Maximum eosinophil survival occurred at 1 to 3 ng/ml IL-1beta. This effect was also time-dependent and was readily detected in airway smooth muscle cell-conditioned medium after just 3 h of stimulation with IL-1beta (1 ng/ml). It continued to increase before reaching a plateau around 24 h, with no decrease in activity for up to 120 h of stimulation. Conditioned medium from unstimulated airway smooth muscle cells did not enhance eosinophil survival. The survival-enhancing activity was completely inhibited (the concentration that inhibited 50% [IC50] was 6.9 microg/ml) by a polyclonal goat antihuman antibody to granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3-100 microg/ml), but antibodies (10-100 microg/ml) to IL-3 and IL-5, and a normal goat immunoglobulin G control had no effect on the eosinophil survival-enhancing activity. GM-CSF levels in culture medium from smooth muscle cells were markedly increased by IL-1beta and were maximum at 30 ng/ml (0.037 ng/ml/10(6) cells versus 3.561 ng/ml/10(6) cells, unstimulated versus 30 ng/ml IL-1beta). The IL-1 receptor antagonist inhibited both the production of GM-CSF (IC50 19. 1 ng/ml) and the eosinophil survival-enhancing (IC50 53.7 ng/ml) activity stimulated by IL-1beta. Release of GM-CSF elicited by IL-1beta was inhibited by dexamethasone but not by indomethacin. These data indicate that cultured human airway smooth muscle cells stimulated with IL-1beta support eosinophil survival through production of GM-CSF and thus may contribute to the local control of inflammatory cell accumulation in the airways.

Selective enhancement of GM-CSF, TNF-alpha, IL-1 beta and IL-8 production by monocytes and macrophages of asthmatic subjects.

Previous work has demonstrated an increase in the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by monocytes derived from asthmatic individuals. We have suggested that monocytes and macrophages enhance airways inflammation by augmented cytokine production. We tested this hypothesis by measuring the production of GM-CSF and macrophage-derived cytokines, namely interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8), from unstimulated and lipopolysaccharide (LPS)-stimulated peripheral blood monocytes and alveolar macrophages in 31 asthmatic and 11 normal, control subjects. The basal production of GM-CSF was four fold higher in the monocytes of asthmatic individuals, but there was no significant difference in the basal production of TNF-alpha, IL-1 beta and IL-8. After stimulation with LPS, asthmatic monocytes produced twofold more GM-CSF and fourfold more IL-1 beta than the monocytes from control subjects. Unstimulated macrophages from asthmatic subjects produced significantly less GM-CSF and TNF-alpha than macrophages from controls, and there was no difference in either IL-1 beta or IL-8 production. When stimulated by LPS, macrophages from asthmatic subjects produced twofold more GM-CSF, threefold more TNF-alpha and fourfold more IL-8. The levels of IL-8 produced by both monocytes and macrophages were at least 20 fold higher than those of the other cytokines measured. There is selectivity in the upregulation of cytokine production by monocytes and macrophages in asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymic synthesis, chemical characterisation and Sda activity of GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc.

The tetrasaccharides GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3'-sialyllactose (NeuAc alpha 2-3 Gal beta 1-4Glc) and 3'-sialyl-N-acetyllactosamine (NeuAc alpha 2-3Gal beta 1-4GlcNAc). The structures of the products were established by methylation and 1H-500 MHz NMR spectroscopy. In Sda serological tests the product formed with 3'-sialyl-N-acetyllactosamine was highly active whereas that formed with 3'-sialyllactose had only weak activity.

A blood group Sda-active pentasaccharide isolated from Tamm-Horsfall urinary glycoprotein.

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal