Intestinal guard: Human CXCL17 modulates protective response against mycotoxins and CXCL17-mimetic peptides development.

Mycotoxin contamination is an ongoing and growing issue that can create health risks and even cause death. Unfortunately, there is currently a lack of specific therapy against mycotoxins with few side effects. On the other hand, the strategic expression of CXCL17 in mucosal tissues suggests that it may be involved in immune response when exposed to mycotoxins, but the exact role of CXCL17 remains largely unknown. Using Caco-2 as a cell model of the intestinal epithelial barrier (the first line of defense against mycotoxins), we showed that a strong production of ROS-dependent CXCL17 was triggered by mycotoxins via p38 and JNK pathways. Under the mycotoxins stress, CXCL17 modulated enhanced immuno-protective response with a remission of inflammation and apoptosis through PI3K/AKT/mTOR. Based on our observed feedback of CXCL17 to the mycotoxins, we developed the CXCL17-mimetic peptides in silico (CX1 and CX2) that possessed the safety and the capability to ameliorate mycotoxins-inducible inflammation and apoptosis. In this study, the identification of detoxifying feature of CXCL17 is a prominent addition to the chemokine field, pointing out a new direction for curing the mycotoxins-caused damage.

Efficient one-pot synthesis of CXCL14 and its derivative using an N-sulfanylethylanilide peptide as a peptide thioester equivalent and their biological evaluation.

CXCL14 is a CXC-type chemokine that exhibits chemotactic activity for immature dendritic cells, activated macrophages, and activated natural killer cells. However, its specific receptor and signaling pathway remain obscure. Recently, it was reported that CXCL14 binds to CXCR4 with high affinity and inhibits CXCL12-mediated chemotaxis. Furthermore, the CXCL14 C-terminal α-helical region is important for binding to its receptor. In this context, we chemically synthesized CXCL14 and its derivative with a one-pot method using N-sulfanylethylanilide peptide as a thioester equivalent. The synthetic CXCL14 proteins possessed inhibitory activities to CXCL12-mediated chemotaxis comparable with that of recombinant CXCL14. Moreover, we proved that chemically biotinylated CXCL14 binds to CXCR4 on cells by flow cytometry analysis.

Synthetic chemokines directly labeled with a fluorescent dye as tools for studying chemokine and chemokine receptor interactions.

Chemokines constitute a protein family that exhibit a variety of biological activities involved in normal and pathological physiological processes. CCL11 (eotaxin), CCL19 (MIP-3beta), CCL22 (MDC), CXCL11 (I-TAC) and CXCL12 (SDF-1alpha) chemokines, modified with the Alexa Fluor 647 fluorescent dye at specific positions along their sequence, were produced by a chemical route and their biological activities were characterized. In a migration assay, fluorescent chemokines were as biologically active as the unmodified forms. All labeled chemokines specifically stained cell lines transfected with the appropriate human chemokine receptors. The specificity of binding was further established by showing that the unlabeled ligands efficiently competed with the labeled chemokines for binding to their respective receptor. A low molecular weight antagonist of CXCR4 prevented binding of labeled CXCL12 to CXCR4 comparably to a neutralizing anti-CXCR4 antibody. Finally, labeled CCL19 was used for the staining of primary cells, illustrating that this reagent can be used for studying CCR7 expression on different cell types. Together, these results demonstrate that fluorescent synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on intact cells, for applications such as cell-based, high throughput screening, and studies of chemokine receptor expression by primary cells.

[Development of selective antagonists against an HIV second receptor].

The authors have discovered a highly selective CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II), and its shortened potent analogs, T140 and TC14012, which strongly inhibit the T-cell line-tropic HIV-1 (X4-HIV-1) infection through their specific binding to a chemokine receptor, CXCR4. CXCR4 is a major coreceptor (second receptor) for the entry of X4-HIV-1 into T-cells. These peptides have been found through the structure-activity relationship (SAR) study on tachyplesins and polyphemusins, which function as self-defense peptides of horseshoe crabs with immature immune systems. T140 and TC14012 showed the highest level of anti-HIV activity and antagonism of target cell entry by X4-HIV-1 among all the CXCR4 antagonists that have been reported to date. Additionally, bifunctional anti-HIV agents based on the specific CXCR4 antagonists (T140 analogs)-3'-azido-3'-deoxythymidine (AZT) conjugation have been synthesized and evaluated, since T140 analogs can possibly work as a carrier of AZT targeting T-cells due to their specific affinity for CXCR4 on T-cells. T22 have two disulfide bonds and a Trp residue in the molecule. In connection with this study, novel facile and side-reaction-free methodologies for disulfide bond formation have been established for the increase of the efficiency of SAR studies. Furthermore, the completely stereocontrolled synthetic process for a couple of (E)-alkene dipeptide isosteres starting from L-amino acid has been established in order to facilitate nonpeptidylation studies on peptide-lead candidates. In this review, the authors wish to summarize our recent research on the development of specific antagonists against the HIV second receptor CXCR4, involving studies on the establishment of efficient methodologies for the facile synthesis of peptides and peptide mimetics.

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)

Identification of a T cell chemotactic factor in the cerebrospinal fluid of HIV-1-infected individuals as interferon-gamma inducible protein 10.

Central nervous system (CNS) involvement is a prominent feature of human immunodeficiency virus (HIV-1) infection. Monocytes and CD4+ T cells traverse the blood brain barrier (BBB), and serve as vehicles for the virus and perpetrators for brain pathology by their production of neurotoxins. In the present study cerebrospinal fluid (CSF) samples from HIV-1-infected patients were analyzed for the presence of chemotactic factors. All 36 CSF samples from the patients were positive for the CXC chemokine interferon-gamma inducible protein (IP-10), which was not detected in CSF samples of 14 controls. The IP-10 concentrations were higher in HIV-1-infected patients with HIV-1 associated neurologic disorders than in those without neurological deficits. In contrast to IP-10, other chemotactic factors including the CC chemokines MCP-1, MIP-1alpha, MIP-1beta and RANTES and the cytokines IL-15 and IL-16 were either not detected or increased in only less than 30% of the patients. Unlike the CSF samples of controls, all CSF samples from HIV-1-infected patients induced chemotaxis of T cells activated with IL-2. The significance of IP-10 as a T cell chemotactic cytokine in HIV-1-infected CSF is shown by (1) the correlation of the IP-10 levels with the extent of T cell chemotaxis, (2) the neutralization of T cell chemotaxis by anti-IP-10 antibodies and (3) the correlation of the chemotactic response of CSF samples on activated T cells and the CSF white cell count in the patients. Our data provide evidence that IP-10 contributes to the accumulation of activated T cells in the CSF compartment in HIV-1-infected individuals.

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.