Faecal calprotectin as reliable non-invasive marker to assess the severity of mucosal inflammation in children with inflammatory bowel disease.

BACKGROUND: An accurate monitoring of mucosal inflammation is important for an effective management of patients with inflammatory bowel disease. Intestinal inflammation can be detected by faecal calprotectin level determination. AIM: To comparatively evaluate the accuracy of faecal calprotectin, clinical scores, common serum markers and endoscopy in the assessment of the severity of intestinal mucosa inflammation in children with inflammatory bowel disease. METHODS: Fifty-eight paediatric patients (mean age 13.9 years, 95% CI 2.9-14.8; male 28) with confirmed inflammatory bowel disease (26 Crohn's disease, 32 ulcerative colitis) were enrolled. Before endoscopy, all patients underwent a complete evaluation including: clinical scores, erythrocyte sedimentation rate, C-reactive protein and faecal calprotectin determination. The severity of mucosal inflammation was assessed using specific endoscopic and histologic scores. RESULTS: Faecal calprotectin showed a high correlation (r=0.655) with the histologic grade of mucosal inflammation, similar to that observed for endoscopy (r=0.699), and it resulted the most accurate tool (sensitivity 94%, specificity 64%, positive predictive value 81%, negative predictive value 87%) to detect the presence of active mucosal inflammation when compared to clinical scores and common serum markers. In patients with apparent clinical and laboratory remission the accuracy of faecal calprotectin resulted further improved (sensitivity 100%, specificity 80%, positive predictive value 67%, negative predictive value 100%). CONCLUSIONS: A more accurate assessment of the severity of mucosal inflammation can be achieved by the determination of faecal calprotectin levels compared to other common clinical and laboratory indices. This non-invasive and objective method could be particular useful in patients with apparent clinical and laboratory remission.

Presentation of chemokine SDF-1 alpha by fibronectin mediates directed migration of T cells.

The role of chemokine-matrix interactions in integrin-dependent T-cell migration was examined to address the critical question of how chemokines provide directional information. The chemokine SDF-1 alpha binds fibronectin (Fn) with a low nanomolar K(d) (equilibrium dissociation constant). SDF-1 alpha presented by Fn induced directed migration. Spatial concentration gradients of chemokine were not required to maintain directed migration. Fn-presented chemokine induced the polarization of cells, including the redistribution of the SDF-1 alpha receptor, to the basal surface and leading edge of the cell. A new model for directed migration is proposed in which the co-presentation of an adhesive matrix and chemokine provides the necessary positional information independent of a soluble spatial gradient. (Blood. 2000;96:2682-2690)

An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster.

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identification of all such gene clusters in a genome. The approach involves sequencing small fragments of a random genomic DNA library containing one or more modular PKS gene clusters, and identifying which fragments emanate from PKS genes. Knowing the approximate sizes of the genome and the target gene cluster, one can predict the the frequency that a PKS gene fragment will be present in the library sequenced. Computer simulations of the approach were applied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clusters in the Bacillus subtilus genome. The approach was then used to identify PKS gene fragments in a strain of Sorangium cellulosum that produces epothilone. In addition to identifying fragments of the epothilone gene cluster, we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrating that the approach is also applicable for identification of these modular gene clusters.

Development and screening of a polyketide virtual library for drug leads against a motilide pharmacophore.

A virtual library of macrocyclic polyketide molecules was generated and screened to identify novel, conformationally constrained potential motilin receptor agonists ("motilides"). A motilide pharmacophore model was generated from the potent 6,9-enol ether erythromycin and known derivatives from the literature. The pharmacophore for each molecular conformation was a point in a distance-volume space based on presentation of the putative binding moieties. Two methods, one fragment based method and the other reaction based, were explored for constructing the polyketide virtual library. First, a virtual library was assembled from monomeric fragments using the CHORTLES language. Second, the virtual library was assembled by the in silico application of all possible polyketide synthase enzyme reactions to generate the product library. Each library was converted to low-energy 3D conformations by distance geometry and standard minimization methods. The distance-volume metric was calculated for low-energy conformations of the members of the virtual polyketide library and screened against the enol ether pharmacophore. The goal was to identify novel macrocycles that satisfy the pharmacophore. We identified three conformationally constrained, novel polyketide series that have low-energy conformations satisfying the distance-volume constraints of the motilide pharmacophore.

Combinatorial discovery process yields antimicrobial peptoids.

N-alkylated glycine trimers are generically referred to as peptoids. The identification of antimicrobial peptoids from a statistically unbiased diverse combinatorial chemistry library led to the design of the optimization peptoid library that we describe in this manuscript. This optimization library was designed using structural information from the most active peptoids in the unbiased library. Screening of the optimization library for antimicrobial activity identified a single pool of peptoids with activity against both Staphylococcus aureus and Escherichia coli. The active peptoids from this pool were active against drug sensitive and drug resistant organisms and represent novel antibacterial compounds.

Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.

BACKGROUND: Fractalkine is a newly identified T-cell and monocyte/macrophage (Mphi) chemokine with a transmembrane domain and is a cell-surface protein on activated endothelium. It can mediate adhesion of cells expressing the fractalkine receptor CX3CR1. These unique features make fractalkine well suited for leukocyte recruitment in tissues with high blood flow as in the renal glomerulus. METHODS: Fractalkine expression in glomeruli and response of isolated glomerular inflammatory cells to fractalkine were studied in the Wistar-Kyoto (WKY) crescentic glomerulonephritis model. Antibody was used to confirm the proinflammatory role of fractalkine. RESULTS: Fractalkine was markedly induced in the endothelium of nephritic rat glomeruli, and inflammatory leukocytes infiltrating the glomeruli expressed increased levels of CX3CR1. Anti-CX3CR1 antibody treatment dramatically blocked leukocyte infiltration in the glomeruli, prevented crescent formation, and improved renal function. CONCLUSIONS: Fractalkine plays a central role in leukocyte trafficking at the endothelium in the high-flow glomerular circuit and, in turn, implicates CX3CR1 as a prime drug target for therapeutic intervention of endothelium-related inflammatory diseases.

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II.

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1alpha, a potent ligand for the HIV-1 "fusin" coreceptor.

Stromal cell-derived factor-1alpha (SDF-1alpha ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1alpha antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1alpha ([N33A]SDF-1alpha ) prepared by total chemical synthesis has been refined to 2.2-A resolution. Although SDF-1alpha adopts a typical chemokine beta-beta-beta-alpha topology, the packing of the alpha-helix against the beta-sheet is strikingly different. Comparison of SDF-1alpha with other chemokine structures confirms the hypothesis that SDF-1alpha may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1alpha reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1alpha with interhelical sites of the receptor, resulting in a biological response.

6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7.

The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.

Chemokines and the arrest of lymphocytes rolling under flow conditions.

Circulating lymphocytes are recruited from the blood to the tissue by rolling along the endothelium until being stopped by a signaling event linked to the Gialpha subunit of a heterotrimeric GTP-binding protein; that event then triggers rapid integrin-dependent adhesion. Four chemokines are now shown to induce such adhesion to intercellular adhesion molecule-1 and to induce arrest of rolling cells within 1 second under flow conditions similar to those of blood. SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2), and MIP-3beta (also called ELC or Exodus-3) induced adhesion of most circulating lymphocytes, including most CD4+ T cells; and MIP-3alpha (also called LARC or Exodus-1) triggered adhesion of memory, but not naïve, CD4+ T cells. Thus, chemokines can regulate the arrest of lymphocyte subsets under flowing conditions, which may allow them to control lymphocyte-endothelial cell recognition and lymphocyte recruitment in vivo.

Genetic subtype-independent inhibition of human immunodeficiency virus type 1 replication by CC and CXC chemokines.

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.

Accessibility of selenomethionine proteins by total chemical synthesis: structural studies of human herpesvirus-8 MIP-II.

The determination of high resolution three-dimensional structures by X-ray crystallography or nuclear magnetic resonance (NMR) is a time-consuming process. Here we describe an approach to circumvent the cloning and expression of a recombinant protein as well as screening for heavy atom derivatives. The selenomethionine-modified chemokine macrophage inflammatory protein-II (MIP-II) from human herpesvirus-8 has been produced by total chemical synthesis, crystallized, and characterized by NMR. The protein has a secondary structure typical of other chemokines and forms a monomer in solution. These results indicate that total chemical synthesis can be used to accelerate the determination of three-dimensional structures of new proteins identified in genome programs.

Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

Chemically synthesized SDF-1alpha analogue, N33A, is a potent chemotactic agent for CXCR4/Fusin/LESTR-expressing human leukocytes.

Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1alpha analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1alpha for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1alpha for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1alpha analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1alpha to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS.

Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines.

Unique among known human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) encodes chemokine-like proteins (vMIP-I and vMIP-II). vMIP-II was shown to block infection of human immunodeficiency virus-type 1 (HIV-1) on a CD4-positive cell line expressing CCR3 and to a lesser extent on one expressing CCR5, whereas both vMIP-I and vMIP-II partially inhibited HIV infection of peripheral blood mononuclear cells. Like eotaxin, vMIP-II activated and chemoattracted human eosinophils by way of CCR3. vMIP-I and vMIP-II, but not cellular MIP-1alpha or RANTES, were highly angiogenic in the chorioallantoic assay, suggesting a possible pathogenic role in Kaposi's sarcoma.

Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.

CHORTLES: a method for representing oligomeric and template-based mixtures.

Screening mixtures of synthetic oligomers or fixed templates (e.g., rings) with varying substituents is increasingly the focus of drug discovery programs. CHORTLES is designed and implemented to facilitate representation, storage, and searching of oligomeric and template-based mixtures of any size. Building upon the CHUCKLES method of representing oligomers as both monomer-based sequences and all-atom structures, CHORTLES compactly represents a mixture without explicitly enumerating individual molecules. This method lends itself to a hierarchy relating mixtures to submixtures and individual compounds, as one finds when deconvoluting mixtures in drug lead discovery programs. In addition, we describe two methods of searching mixtures at the monomer level. We also present a simple pictorial representation for describing all components in a mixture, which becomes essential as the list of monomer names is expanded beyond common names (e.g., amino acids).

Measuring diversity: experimental design of combinatorial libraries for drug discovery.

Screening synthetic combinatorial libraries, such as mixtures of oligo(N-substituted)glycines, facilitates rapid drug lead discovery and optimization by vastly increasing the number of candidate molecules made and tested. Discovery efficiency and productivity can be further improved by using experimental design to maximize molecular diversity for a given library size or to bias the library with key features for a specific receptor. We describe new methods to quantify molecular diversity using descriptors that characterize lipophilicity, shape and branching, chemical functionality, and specific binding features. Experimental design methods select sets of side chains that are diverse in these properties, and "flower plots" allow the diversity to be graphically compared. We also quantify the overall diversity accessible to different families of combinatorial chemistry.

Evaluation of breast stimulation for induction of labor in women with a prior cesarean section and in grandmultiparas.

BACKGROUND: Information on the efficacy of breast stimulation for inducing labor in grandmultiparas and in women with a previous cesarean section with or without premature rupture of membranes is limited. METHODS: Retrospective study of labor data from 135 women of grand multiparity or those with a previous section with or without premature rupture of the membranes in whom labor was induced by breast stimulation. RESULTS: The success rate in achieving vaginal delivery was 84%. The duration of breast stimulation, length of labor, vaginal delivery rate, and Apgar score did not differ significantly among the four groups studied. CONCLUSION: Breast stimulation in grandmultiparas and in women with a previous cesarean section is efficacious and safe.