Separating cytokine twins with a small molecule.

The cytokine macrophage migration inhibitory factor (MIF) has been characterized as a key immunomodulator and mediator of various diseases. Small molecule inhibitors based on the conserved enzymatic pocket of MIF have been valuable in elucidating MIF mechanisms and developing translational strategies. In contrast, our mechanistic understanding of the MIF homolog MIF-2/d-dopachrome tautomerase (d-DT) and its clinical translation has been hampered, partly because MIF-2-selective inhibitors have been elusive. Here, Tilstam et al. characterize a small-molecule inhibitor selective for MIF-2 that interferes with receptor binding and cell signaling. That could be a promising therapeutic lead and a valuable research tool.

Progress in pharmacological research of chemokine like factor 1 (CKLF1).

Currently, the research of chemokines has penetrated into many fields of life science. A new kind of chemokines, chemokine like factor 1 (CKLF1), which is cloned through suppression subtractive hybridisation (SSH) technology is expressed widely in human body, especially in the lung and peripheral blood leukocytes. CKLF1 has a broad spectrum of chemotaxic activity for many cells, such as lymphocytes, macrophages, bone marrow cells, nerve cells and so on. In addition, CKLF1 also stimulates the regeneration of skeletal muscle cells in vivo. Collecting data derived from our and other laboratories show that CKLF1 has an important relationship with allergic diseases, autoimmune diseases, tumors, cardio-cerebrovascular diseases and so on. Therefore, there be an important theoretical purport and applied value to make a summary of pharmacological progress of CKLF1.

Nanotraps with biomimetic surface as decoys for chemokines.

A creation of nanotraps that could selectively recognize the chemotactic mediators of leukocyte adhesion and eliminate them from the bloodstream and tissue intercellular matrix is a promising approach for the treatment of various inflammatory and autoimmune diseases. We designed nanotraps as artificial decoy receptors based on poly(lactic acid) (PLA) nanoparticles covered by heparin and bearing on the surface two fragments of CCR5 receptor (N-terminal domain, Nt, and second extracellular loop, ECL2), responsible for chemokine binding. In order to attach Nt and ECL2 to the heparin shell, the corresponding peptides were modified with N- and/or C-terminal oligolysines. The presence of the nanotraps in the cell medium completely eliminated the activating effect of a CCR5 ligand, chemokine Rantes, while strongly decreasing the adhesion of monocytes to the human endothelial cells. We found that the modified ECL2 alone was also able to prevent monocyte adhesion, thus acting as a decoy receptor itself.

Pregnant women infected with pandemic influenza A(H1N1)pdm09 virus showed differential immune response correlated with disease severity.

BACKGROUND: During pregnancy, immunological and hormonal alterations place women at increased risk for influenza-related severe illnesses including hospitalization and death. Although A(H1N1) pdm09 infection resulted in increased disease severity in pregnant women, the precise mechanisms responsible for this risk have yet to be established. OBJECTIVES: The present study was aimed to investigate the role of host chemokines and cytokine profiles in A(H1N1) pdm09 infection regarding disease severity in pregnant women. STUDY DESIGN: This retrospective survey examined 41 pregnant women with confirmed A(H1N1) pdm09 infection. Of them, 12 died (D), 29 survived (S), and 17 remained uninfected and served as controls (C). Antiviral response was evaluated for IFN-ß expression and gene expression profiles of cytokines (TNF-α, IL-6, IL-12, TGF-ß) and chemokines (IL-8, RANTES, MCP-1, IP-10), and the viral Matrix (M1) gene was quantified and normalized using the housekeeping gene product ß-actin mRNA. RESULTS: Higher IL-8 and TNF-α mRNA expression were found in D and S compared with C, while IL-6 showed higher expression in D. Interestingly, these results were associated with a decrease in the anti-inflammatory response of TGF-ß mRNA and IFN-ß. These alterations would lead to an imbalance in the immune response of those patients. CONCLUSIONS: Pregnancy-related reductions in IFN-ß and TGF-ß expression levels and elevated levels of pro-inflammatory cytokines could explain the increased severity of infection and death of pregnant women. These findings may help improve the understanding of the high susceptibility and disease severity to influenza virus infection during pregnancy.

Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties.

Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochemistry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoattractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-α, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.

A biologically active vMIP-II-IgG3-TfN fusion protein, secreted from methylotrophic yeast Pichia pastoris.

The viral macrophage inflammatory protein II (vMIP-II) which showed a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus. To explore the feasibility of using TfN as a carrier moiety for delivery of therapeutic proteins, a genetically engineered vMIP-II-IgG3-TfN fusion gene was loaded into the yeast expression vector pPICZα. The linearized recombinant plasmid pPICZα-vMIP-II-IgG3-TfN was transformed into X33 competent cells. The recombinant protein was expressed in methylotrophic yeast Pichia pastoris and was confirmed to have expected molecular mass of 48 kDa by SDS-PAGE. Using methods combining ammonium sulfate precipitation, dialysis, ultrafiltration and affinity chromatography, the vMIP-II-IgG3-TfN fusion protein was successfully purified from the supernatant of the broth. Western-blotting analysis showed that 6× His antibody recognized the purified vMIP-II-IgG3-TfN. CD spectrum revealed a positive peak at 196.5 nm and a negative peak at 209 nm. MALDI-TOF MS analysis showed that the purified vMIP-II-IgG3-TfN was an intact and homogeneous protein. The pepsin digestibility assay showed that the vMIP-II-IgG3-TfN fusion protein could be digested into small fragments by pepsin after 2 min treatment. The vMIP-II-IgG3-TfN fusion protein was found to be stable in human plasma for up to 48 h. Furthermore, in vitro bioactivity assay indicated that the vMIP-II-IgG3-TfN fusion protein can block the chemotaxis of U937 cells induced by SDF1α. In total, this study illustrates the development of an active vMIP-II-IgG3-TfN fusion protein expressed in P. pastoris.

3D profile-based approach to proteome-wide discovery of novel human chemokines.

Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D) structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides, the genome-wide applicability of our methodology based on 3D protein family profiles may open up new possibilities for improving and accelerating protein function annotation processes.

Efficacy of an enterovaccine in recurrent episodes of diarrhea in the dog: a pilot study

Recurrent episodes of self-limiting diarrhea in the dog, due to sudden dietary changes and to stressful or exciting situations, are conditions sometimes difficult to treat. Colifagina®, a commercially available bacterial enterovaccine, showed, in previous studies performed on experimentally induced colitis in mice, to be able to improve both disease activity index and histological appearance, increase colonic secretion of IgA, and reduce inflammatory chemokine secretion. In the present study Colifagina® was administered to five dogs presenting recurrent episodes of self-limiting diarrhea and to one dog presenting chronic diarrhea. During the follow-up period, almost all patients decreased the number of episodes of abnormal defecation and the fecal score of such episodes improved in five out of six dogs. Even if further studies are needed to understand the exact potential of the compound, in dogs presenting recurrent episodes of self-limiting diarrhea due to sudden dietary changes and/or stressing or exciting situations, Colifagina® seems to be helpful in managing most of these patients(AU)

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies.

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.

Blood purification in sepsis: a new paradigm.

Sepsis is one of the main causes of death in critically ill patients. The pathophysiology of sepsis is complex and not completely understood. The proinflammatory and anti-inflammatory response leads to cell and organ dysfunction and, in many cases, death. Thus, the goal of the intervention is to restore the homeostasis of circulating mediators rather than to inhibit selectively the proinflammatory or anti-inflammatory mediators. Blood purification has been reported to remove a wide array of inflammatory mediators. The effects are broad-spectrum and auto-regulating. Blood purification has also been demonstrated to restore immune function through improving antigen-presenting capability, adjusting leukocyte recruitment, oxidative burst and phagocytosis, and improving leukocyte responsiveness. A great deal of work has to be done in order to find and optimize the best extracorporeal blood purification therapy for sepsis. New devices specifically target the pathophysiological mechanisms involved in these conditions. High-volume hemofiltration, hemoadsorption, coupled plasma filtration adsorption, and high cutoff membrane are now being tested in septic patients. Preliminary data indicate the feasibility of these modified techniques in sepsis. Their impact on patient prognosis, however, still needs proof by large randomized clinical trials. Finally, the emerging paradigm of sepsis-induced immune suppression provides additional rationale for the development of extracorporeal blood purification therapy for sepsis.

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli.

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/microg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.

Chapter 1. Isolation, identification, and production of posttranslationally modified chemokines.

Chemokines attract cells during the development of lymphoid tissues, leukocyte homing, and pathologic processes such as cancer and inflammation. Limited posttranslational modification of chemokines may significantly alter the glycosaminoglycan and/or receptor binding properties and signaling potency of these chemotactic proteins. To compare the in vitro and in vivo biologic activities of posttranslationally modified chemokine isoforms, considerable amounts of pure chemokine isoforms are required. This chapter describes a number of chromatographic techniques that are useful for the isolation of natural, posttranslationally modified chemokines from primary human cell cultures. In addition, combination of immunologic assays and biochemical techniques such as automated Edman degradation and mass spectrometry are used for the identification of modifications. Alternate methods for the generation of specific chemokine isoforms are discussed such as modification of chemokines by specific enzymes and total chemical syntheses and folding of chemokine isoforms. In particular, in vitro processing of chemokines by the protease aminopeptidase N/CD13 and citrullination or deamination of chemokines by peptidyl arginine deiminases (PAD) are described as methods for the confirmation or generation of posttranslationally modified chemokine isoforms.

Optimized procedures for producing biologically active chemokines.

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.

Afferent and efferent interfaces of lymph nodes are distinguished by expression of lymphatic endothelial markers and chemokines.

BACKGROUND: Lymph nodes (LNs) are important sites of connection between the sampled peripheral tissues, the many cells of the immune system, and the blood. The organization of the interface between the afferent and efferent lymphatic vasculature and LN parenchyma is incompletely understood, and obtaining a better understanding of these tissue microenvironments will contribute to an improved understanding of overall lymphatic function. METHODS AND RESULTS: We used histologic approaches to define the distributions of cells expressing lymphatic endothelial cell (LEC) markers in LNs from healthy, simian immunodeficiency virus (SIV) infected, or Mycobacterium tuberculosis infected cynomolgus macaques. Cells at the afferent and efferent interfaces of LNs from all animals showed differential expression of LEC markers, with podoplanin, Prox-1, and VEGFR3 expressed in both microenvironments, but with LYVE-1 expressed only at the efferent interface. The chemokine CCL20 was uniquely expressed at the afferent interface by cells co-expressing podoplanin, and this expression was increased during SIV or M. tuberculosis infection. In contrast, only a small proportion of cells expressing the CCR7 ligand CCL21 co-expressed podoplanin. Treatment of model LECs with the TLR3 ligand poly(I:C) or gamma-irradiated M. tuberculosis increased production of CCL20 without altering CCL21 or LEC marker expression. CONCLUSIONS: This study provides a comprehensive mapping of the organization of the lymphatic endothelial network entering and exiting LNs in health and in chronic infectious diseases in a nonhuman primate model. The differences we have defined between the afferent and efferent interfaces of LNs could inform the future design of vaccines and immunotherapies.

VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism.

BACKGROUND: The lymphatic endothelium is an important semi-permeable barrier separating lymph from the interstitial space. However, there is currently a limited understanding of the lymphatic endothelial barrier and the mechanisms of lymph formation. The objectives of this study were to investigate the potential active role of lymphatic endothelial cells in barrier regulation, and to test whether the endothelial cell agonists VEGF-A and VEGF-C can alter lymphatic endothelial barrier function. METHODS AND RESULTS: Cultured adult human dermal microlymphatic endothelial cells (HMLEC-d) and human umbilical vein endothelial cells (HUVEC) were respectively used as models of lymphatic and vascular endothelium. Transendothelial electrical resistance (TER) of endothelial monolayers served as an index of barrier function. Cells were treated with VEGF-A, VEGF-C, or the VEGFR-3 selective mutant VEGF-C156S. MAZ51 was used to inhibit VEGFR-3 signaling. The results show that while VEGF-A causes a time-dependent decrease in TER in HUVEC, there is no response in HMLEC-d. In contrast, VEGF-C and VEGF-C156S cause a similar decrease in TER in HMLEC-d that is not observed in HUVEC. These results corresponded to the protein expression of VEGFR-2 and VEGFR-3 in these cell types, determined by Western blotting. In addition, the VEGF-C- and VEGF-C156S-induced TER changes were inhibited by MAZ51. CONCLUSIONS: The results indicate differential responses of the lymphatic and vascular endothelial barriers to VEGF-A and VEGF-C. Furthermore, our data suggest that VEGF-C alters lymphatic endothelial function through a mechanism involving VEGFR-3.

Influence of interleukin-2 deficiency on the generation of autoimmune B cells.

The production of auto-antibodies is one of the predominant characteristics of autoimmune disorders. Because IL-2 deficient mice develop autoimmunity, we asked how IL-2 deficiency might impair endogenous mechanisms of B cell tolerance. To this end, we mated BALB/c anti-dsDNA H chain knock-in mice, in which B cells producing anti-dsDNA antibodies are properly regulated, with IL-2 deficient mice and assessed the phenotype of their offspring. IL-2 deficient mice expressing the anti-dsDNA H chain knock-in allele developed anti-dsDNA antibodies of both IgM and IgG isotypes. Production of these antibodies occurred through the disruption of several mechanisms of endogenous tolerance, including deletion, maturational arrest, and follicular exclusion. In summary, our results suggest that IL-2 plays an important role in regulating B cell tolerance.

On-column refolding of recombinant chemokines for NMR studies and biological assays.

We have applied an efficient solid-phase protein refolding method to the milligram scale production of natively folded recombinant chemokine proteins. Chemokines are intensely studied proteins because of their roles in immune system regulation, response to inflammation, fetal development, and numerous disease states including, but not limited to, HIV-1/AIDS, cancer metastasis, Crohn's disease, asthma and arthritis. Many investigators use recombinant chemokines for research purposes, however these proteins partition almost exclusively to the inclusion body fraction when produced in Escherichia coli. A major hurdle is to correctly refold the chemokine and oxidize the two highly conserved disulfide bonds found in nearly all chemokines. Conventional methods for oxidation and refolding by dialysis or extreme dilution are effective but slow and yield large volumes of dilute chemokine. Here we use an on-column approach for rapid refolding and oxidation of four chemokines, CXCL12/SDF-1alpha (stromal cell-derived factor-1alpha), CCL5/RANTES, XCL1/lymphotactin, and CX3CL1/fractalkine. NMR spectra of SDF-1alpha, RANTES, lymphotactin, and fractalkine indicate these chemokines adopt native structures. On-column refolded SDF-1alpha is fully active in an intracellular calcium flux assay. Our success with multiple SDF-1alpha mutants and members of all four chemokine subfamilies suggests that on-column refolding is a robust method for preparative-scale production of recombinant chemokine proteins.

In vitro and in vivo protein sampling by combined microdialysis and ultrafiltration.

Cytokines, chemokines and growth factors regulate inflammation, resistance to infection and tissue repair. Understanding their function within tissues is a priority in evolving therapy for a number of disease processes. Yet, the existence of complex networks of these factors in the tissue microenvironment has made understanding of their interactions difficult. We demonstrate the capability of microdialysis probes to recover small proteins efficiently in vitro. Further we show that microdialysis of human tissues allows for protein recovery from tissue interstitial fluid. This technology, combined with a multiplexed immunoassay, facilitates the simultaneous measurement of cytokines and chemokines in response to injury in the oral mucosa of human subjects in vivo.

Oral administration of second-generation immunomodulatory oligonucleotides induces mucosal Th1 immune responses and adjuvant activity.

CpG DNA induces potent Th1 immune responses through Toll-like receptor 9. In the present study, we used oligonucleotides consisting of a novel 3'-3'-linked structure and synthetic stimulatory motifs, referred as second-generation immunomodulatory oligonucleotides (IMOs). The stimulatory motifs included: CpR, YpG, or R'pG (R = 2'-deoxy-7-deazaguanosine, Y = 2'-deoxy-5-hydroxy-cytidine, and R' = 1-[2'-deoxy-beta-d-ribofuranosyl]-2-oxo-7-deaza-8-methyl-purine). We evaluated the stability of orally administered IMOs in the gastrointestinal (GI) environment and their ability to induce mucosal immune responses in mice, and compared these characteristics with those of a conventional CpG DNA. The IMOs were significantly more stable than CpG DNA following oral administration, and IMOs induced stronger local and systemic immune responses as determined by MIP-1beta, MCP-1, IP-10, and IL-12 production. Mice orally immunized with ovalbumin (OVA) and IMO had higher levels of IgG2a antibodies in serum and IgA antibodies in intestinal mucosa than did mice immunized with OVA and CpG DNA. These studies demonstrate that IMOs are more stable than CpG DNA in the GI tract and can induce more potent mucosal Th1 adjuvant responses. IMOs may prove to be effective oral adjuvants, able to promote strong systemic and mucosal immune responses to oral vaccines and antigens for therapeutic and prophylactic applications.