Magnetic bead based assays for complement component C5.

Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N'-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5.

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma.

Complement activation in the context of stem cells and tissue repair.

The complement pathway is best known for its role in immune surveillance and inflammation. However, its ability of opsonizing and removing not only pathogens, but also necrotic and apoptotic cells, is a phylogenetically ancient means of initiating tissue repair. The means and mechanisms of complement-mediated tissue repair are discussed in this review.

Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex.

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains.

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC).

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains.

The chemokine CCL18 causes maturation of cultured monocytes to macrophages in the M2 spectrum.

The observation that human monocytes cultured in the presence of the chemokine CCL18 showed increased survival, led us to profile cytokine expression in CCL18-stimulated versus control cultures. CCL18 caused significantly increased expression of chemokines (CXCL8, CCL2, CCL3 and CCL22), interleukin-10 (IL-10) and platelet-derived growth factor, but no up-regulation of M1 cytokines IL-1β or IL-12. CCL18-stimulated monocytes matured into cells with morphological resemblance to IL-4-stimulated macrophages, and expressed the monocyte marker CD14 as well the M2 macrophage markers CD206 and 15-lipoxygenase, but no mature dendritic cell markers (CD80, CD83 or CD86).

Mesenchymal stem cells and their microenvironment.

Mesenchymal stem cells (MSC) are multipotent stem cells that hold promise for an expanding list of therapeutic uses, not only due to their ability to differentiate into all connective tissues including bone, fat and cartilage, but additionally due to their trophic and anti-inflammatory effects which contribute to healing and tissue regeneration. Ongoing research is starting to illuminate important aspects of the microenvironmental niche, which supports MSC self-renewal. In this review, we summarize recent findings on cellular structures and molecular pathways that are involved in regulation of MSC self-renewal versus differentiation, and in retention of MSCs within the niche versus mobilization and recruitment to sites of injury.

Expression of soluble proteins in Escherichia coli by linkage with the acidic propiece of eosinophil major basic protein.

An expression method has been developed to produce soluble cationic polypeptides in Escherichia coli while avoiding inclusion body deposition. For this technique the recombinant product is linked through a thrombin or factor Xa susceptible bond to the amino-terminal domain of the precursor of eosinophil major basic protein (MBP). This N-terminal domain is strongly acidic and is apparently able to shield eosinophils from the potentially injurious activities of MBP.

Alpha 7 subunit of nAChR regulates migration of human mesenchymal stem cells.

The efficient migration of mesenchymal stem cells (MSCs) to diseased tissues is required for the fulfillment of their regenerative potential. Recruitment of circulating cells into the damaged tissues is regulated by a complex network, which includes the non-neural cholinergic system. We found that human MSCs (hMSCs) express nicotinic acetylcholine receptor subunits alpha 7, beta 2 and beta 4.

C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation.

Mesenchymal stem cells (MSCs) have a great potential for tissue repair, especially if they can be delivered efficiently to sites of tissue injury. Since complement activation occurs whenever there is tissue damage, the effects of the complement activation products C3a and C5a on MSCs were examined. Both C3a and C5a were chemoattractants for human bone marrow-derived MSCs, which expressed both the C3a receptor (C3aR) and the C5a receptor (C5aR; CD88) on the cell surface.

The role of the complement anaphylatoxins in the recruitment of eosinophils.

Eosinophils are blood and tissue immune cells that participate in a diverse range of activities normally beneficial for the host defense, but in circumstances of untoward inflammatory conditions these cells can be responsible for pathological responses. Accordingly the transit of eosinophils from the blood to tissues is a subject of considerable importance in immunology. In this article we review how the complement anaphylatoxins, C3a and C5a bring about eosinophil extravasation.

Akt plays an important role in breast cancer cell chemotaxis to CXCL12.

The chemokine receptor CXCR4 is functionally expressed on the cell surface of various cancer cells, and plays a role in cell proliferation and migration of these cells. Specifically, in breast cancer cells the CXCR4/CXCL12 axis has been implicated in cell migration in vitro and in metastasis in vivo, but the underlying signaling mechanisms are incompletely understood. The xenograft-derived MDA-MB-231 breast cancer cell line (231mfp), which was shown previously to grow more aggressively than the parent cells, showed increased CXCR4 expression at the mRNA, total protein and cell surface expression level.

CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes.

Chemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture.

C5a mediates secretion and activation of matrix metalloproteinase 9 from human eosinophils and neutrophils.

Matrix metalloproteinase 9 (MMP-9) is a crucial proteinase, utilized by both eosinophils and neutrophils, that mediates transmigration through extracellular basement membranes. We have found that neutralization of MMP-9 by a monoclonal antibody or a chemical inhibitor blocked C5a dependent chemotaxis of these granulocytes in vitro. The levels of MMP-9 secreted by the action of C5a from eosinophils were about 50-fold lower than those from neutrophils, consistent with results from confocal microscopy, where the density of MMP-9 containing granules was fewer within eosinophils than in neutrophils.

Regulation of CXCR4-mediated nuclear translocation of extracellular signal-related kinases 1 and 2.

Activation of the chemokine receptor CXCR4 by its agonist stromal cell-derived factor 1 (SDF-1) has been associated with cell migration and proliferation in many cell types, but the intracellular signaling cascades are incompletely defined. Here we show that CXCR4-dependent extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation was mediated through the Ras/Raf pathway, as demonstrated with a dominant-negative Ras mutant and pharmacological inhibitors. The Src inhibitor 4-amino-5-methylphenyl-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and the Rho-kinase (ROCK) inhibitor N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y27632) also attenuated SDF-1-induced ERK1/2 phosphorylation.

Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2.

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis.

Eosinophils and monocytes produce pulmonary and activation-regulated chemokine, which activates cultured monocytes/macrophages.

Pulmonary and activation-regulated chemokine (PARC/CCL18) belongs to the family of CC chemokines and shares 61% sequence identity with monocyte inflammatory protein (MIP)-1alpha. Produced by dendritic cells and macrophages primarily in the lung, PARC is known to be chemotactic for T cells. Because PARC's biological function is largely unknown, we screened various leukocyte populations for PARC expression and for response to PARC, with the idea that the cellular source may link PARC to disease states in which it may be involved.

An alternative processing of integrin alpha(v) subunit in tumor cells by membrane type-1 matrix metalloproteinase.

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alpha(v)beta(3) integrin are both essential to cell invasion. Maturation of integrin pro-alpha(v)chain (pro-alpha(v)) involves its cleavage by proprotein convertases (PC) to form the disulfide-bonded 125-kDa heavy and 25-kDa light alpha chains. Our report presents evidence of an alternative pathway of pro-alpha(v) processing involving MT1-MMP.