Seasonal variation in whole blood cytokine production after LPS stimulation in normal individuals

We examined seasonal differences in whole blood cytokine production after endotoxin (LPS) stimulation in 17 healthy individuals from an urban area having normal sleep/wakefulness pattern. We used 500 pg/ml of LPS for incubation period of 4 h to stimulate 100 microl of whole blood of the same subjects in June, September, February, and March. We found no differences in the circulating total WBCs and differentials including monocytes between different seasons. We found during September (autumn) a reduced pro-inflammatory cytokine production in terms of TNF-alpha and IL-6 production compared to the other seasons. We also found a reduced anti-inflammatory cytokine production in June (summer) and September (autumn) in terms of IL-10, TNF-RI and TNF-RII compared to February (winter) and March (spring). Our results suggest that in early summer there is a predominating pro-inflammatory cytokine response which is counterbalanced early in autumn. These results may have significant implications in the determination of reference values, in exploration of immune response and inflammatory disease prevalence between different seasons, in determining LPS tolerance (immunoparalysis) and planning clinical trials and immunomodulary therapies. However, the effect of dark/light exposure differences on the circadian periodicity in the responsiveness of immune cells during different seasons should be further investigated.     

Zymosan-induced luminol-amplified chemiluminescence of whole blood phagocytes in experimental and human hyperthyroidism

Luminol-amplified CL of whole blood phagocytes was studied in rats given 3 consecutive doses of 0.1 mg L-triiodothyronine T₃/kg or in hyperthyroid patients, after stimulation by zymosan. In both cases, CL was significantly increased, in effect which was produced independently of the opsonization of the zymosan particles and markedly inhibited by azide. The in vitro addition of T₃ or L-thyroxine (T₄) to whole blood phagocytes from normal rats did not modify the opsonized zymosan-dependent CL, when assayed at the concentrations found in eutrhyroid subjects or in hyperthyroid patients. Administrations of propylthiouracil (400 mg/day for 2–3 months) to hyperthyroid patients reduced the CL response observed prior to treatment, to values comparable to those found in the euthyroid group. These data indicate that hyperthyroidism elicits an enhanced respiratory burst activity of whole blood phagocytes, probably related to adaptive changes induced by thyroid hormone on the mieloperoxidase-H₂O₂ system, rather than to direct actions of the hormone molecule or changes in the opsonic capacity of plasma.     

Toxic and essential elements in children's blood (<6 years) from Kinshasa,DRC (the Democratic Republic of Congo)

In this study we determined the concentration of 9 trace elements (As, Cd, Cu, Hg, Mn, Mo, Pb, Se and Zn) in whole blood of children (n = 100, 64 girls, 36 boys and median age: 36 months) using inductively coupled plasma mass spectrometry (ICP-MS). The proportion of children potentially deficient in essential elements or poisoned by toxic elements was evaluated. The aging effects on the concentration of these elements were also investigated. The median values were 3.17 μg/L (As), 0.15 μg/L (Cd), 1.1 mg/L (Cu), 2.1 μg/L (Hg), 10.4 μg/L (Mn), 17.7 μg/L (Mo), 8.7 μg/dL (Pb), 10.7 μg/L (Se) and 5.0 mg/L (Zn). The concentration of many elements (As, Cd, Hg, Mn, Pb and Zn) showed significant age variations but not sex influence. Regarding levels of the essential elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except B-Cd, all toxic elements showed exceeded blood levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations.     

Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.     

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.     

Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.     

斜纹夜蛾GNBP1基因的克隆及诱导表达模式的研究

革兰氏阴性结合蛋白(Gram-negative bacteria binding proteins,GNBPs)是昆虫天然免疫系统中的一类重要模式识别受体,在识别病原微生物表面的相关分子模式中发挥着重要作用。为了解模式识别蛋白GNBP如何免疫应答病原微生物应激,本研究利用RT-PCR结合RACE技术克隆获得斜纹夜蛾革兰氏阴性结合蛋白GNBP1基因(SlGNBP1)。斜纹夜蛾SlGNBP1基因(GenBank登录号:JF313110)全长cDNA序列为1 448 bp,开放阅读框(ORF)序列长1 302 bp,编码433个氨基酸残基。生物信息学分析序列表明SlGNBP1蛋白序列含有βGRP(β-1,3-glucan recognition protein)家族特有的保守性结构域,即糖苷水解酶活性结构域(Glyco_hydro_16),有3个N-糖基化位点,19个O-糖基化位点,42个磷酸化位点。系统进化树和同源性分析表明斜纹夜蛾SlGNBP1基因与其它鳞翅目昆虫的βGRP1基因的同源性很高,其中,斜纹夜蛾SlGNBP1基因与棉铃虫和柑橘凤蝶的βGRP序列相似度最高,相似度分别为79%和66%,SlGNBP1蛋白序列中有4个保守的半胱氨酸位点,富含12个色氨酸,由于两个葡聚糖酶活性位点突变而失去葡聚糖酶活性。利用实时荧光定量PCR (RT-qPCR)检测斜纹夜蛾SlGNBP1基因的时空表达模式,结果表明SlGNBP1基因在斜纹夜蛾整个发育历期都表达,其中4龄幼虫表达量最高;组织分布表明SlGNBP1基因在斜纹夜蛾的表皮、脂肪体、中肠、卵巢和血细胞中均有表达,其中脂肪体为主要表达部位。将绿僵菌、玫烟色棒束孢、金黄色葡萄球菌、苏云金芽孢杆菌、大肠杆菌和粘质沙雷氏菌6种病原微生物分别注射4龄斜纹夜蛾幼虫后,对SlGNBP1基因进行mRNA水平检测。RT-qPCR检测表明SlGNBP1在不同诱导时间点与对照相比,均呈现明显上调表达,但诱导程度不同,诱导18-24 h后达到峰值,且SlGNBP1基因对粘质沙雷氏菌最为敏感,与对照相比提高了约100倍。以上结果表明SlGNBP1基因可能在斜纹夜蛾先天免疫应答反应中通过识别病原微生物表面的相关分子模式而发挥作用,可能成为斜纹夜蛾生物防治的新靶标。     

Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface,Promoting Oligomerization,and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.     

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.     

Activation state of alpha4beta1 integrin on sickle red blood cells is linked to the duffy antigen receptor for chemokines (DARC) expression

In sickle cell anemia, reticulocytes express enhanced levels of α4β1 integrin that interact mainly with vascular cell adhesion molecule-1 and fibronectin, promoting vaso-occlusion. These interactions are known to be highly sensitive to the inflammatory chemokine IL-8. The Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes. However, the link between α4β1 activation by chemokines and DARC erythroid expression is not or poorly explored. Therefore, the capacity of α4β1 to mediate Duffy-negative and Duffy-positive sickle reticulocyte (SRe) adhesion to immobilized vascular cell adhesion molecule-1 and fibronectin was evaluated. Using static adhesion assays, we found that, under basal conditions, Duffy-positive SRe adhesion was 2-fold higher than that of Duffy-negative SRes. Incubating the cells with IL-8 or RANTES (regulated on activation normal T cell expressed and secreted) increased Duffy-positive SRe adhesion only, whereas Mn(2+) increased cell adhesion independently of the Duffy phenotype. Flow cytometry analyses performed with anti-β1 and anti-α4 antibodies, including a conformation-sensitive one, in the presence or absence of IL-8, revealed that Duffy-positive and Duffy-negative SRes displayed similar erythroid α4β1 expression levels, but with distinct activation states. IL-8 did not affect α4β1 affinity in Duffy-positive SRes but induced its clustering as corroborated by immunofluorescence microscopy. Our results indicate that in Duffy-negative SRes α4β1 integrin is constitutively expressed in a low affinity state, whereas in Duffy-positive SRes α4β1 is expressed in a higher chemokine-sensitive affinity state. This activation state associated with DARC RBC expression may influence the intensity of the inflammatory responses encountered in sickle cell anemia and participate in its interindividual clinical expression variability.     

Poor induction of interleukin-2 receptor expression on CD8bright+ cells in whole blood cell cultures with CD3 mAb. Implications for immunotherapy with CD3 mAb

To induce better stimulation of T cells during recombinant interleukin-2 (rIL-2) therapy of renal cell carcinoma patients, pretreatment with low-dose CD3 monoclonal antibody (mAb) has been proposed. However, in our clinic, such a treatment did not induce additional activation of T cells. To investigate this we performed whole blood cell cultures with rIL-2 or CD3 mAb as a stimulant. Cultures using isolated blood mononuclear cells were used as a control. When stimulated by the addition of rIL-2, the lymphocyte composition and activation of whole blood cultures did not differ from those of mononuclear cell (MNC) cultures. However, when stimulation was performed with CD3 mAb, CD8bright⁺ cells in whole blood cultures were not or only minimally induced to express CD25 or IL-2 receptor (IL-2R\). This is in contrast to the situation found in MNC cultures where all CD8bright⁺ cells expressed CD25 or IL-2Rß to a high extent at the end of culture. When rIL-2 or recombinant interferon (rIFN) was added to whole blood cultures together with CD3 mAb, significantly more CD8bright⁺ cells were induced to express CD25 or IL-2Rß. These results suggest that whole blood cultures represent the in vivo situation better than MNC cultures. In addition, the results suggest that, also in vivo, administration of low-dose CD3 mAb alone might not be sufficient to induce IL-2R expression on CD8bright⁺ cells, and would therefore not induce additional specific T cell activation in rIL-2-based immunotherapy. The presented results suggest that in vivosimultaneous administration of rIFN or rIL-2 with low-dose CD3 mAb might induce better stimulation of CD8⁺ T cells than CD3 mAb only.     

Orange-spotted grouper (Epinephelus coioides) toll-like receptor 22: molecular characterization, expression pattern and pertinent signaling pathways

The toll-like receptors (TLRs) are an important gene family in host innate immunologic surveillance. The TLR22 gene is an essential member of the TLRs that is only found in aquatic animals and has been detected in some bony fish. Here, a TLR22 homolog, EcTLR22, was characterized in the orange-spotted grouper (Epinephelus coioides) via homology cloning. The 3321 bp full-length cDNA sequence of EcTLR22 was obtained, which included an open reading frame of 2880 bp encoding a putative peptide of 960 amino acids containing three highly typical domains with the characteristics of TLR family members. The deduced amino acid sequence of EcTLR22 showed a relatively high similarity to flounder TLR22. Phylogenetic analysis showed that the orange-spotted grouper TLR22 sequence was clustered with those of Perciforme, such as flounder and croaker. Real-time quantitative PCR analysis revealed broad expression of EcTLR22, with relatively high expression detected in the head kidney, trunk kidney, spleen, peripheral blood leukocytes (PBLs) and heart of orange-spotted grouper. After injection with Vibrio alginolyticus, there was significant up-regulation of the expression of EcTLR22 in the spleen. In evaluating unstimulated/stimulated head kidney leukocytes and spleen leukocytes, a significant increase in EcTLR22 mRNA expression was detected, which implied a sensitive immune response. Furthermore, four important molecules for signal transduction, MyD88, TRIF, TNF-α and IRF3, were chosen to analyze the role of the EcTLR22 signaling pathway in anti-pathogen responses. Upon LPS or Poly I:C challenge, expression of the four genes was induced, with an increasing tendency detected in head kidney leukocytes, suggesting that the four genes might work with EcTLR22 in host defense against pathogenic microbes.     

Induction of C-X-C Chemokine Receptor Type 7 (CXCR7) Switches Stromal Cell-derived Factor-1 (SDF-1) Signaling and Phagocytic Activity in Macrophages Linked to Atherosclerosis

The discovery of CXCR7 as a new receptor for SDF-1 places many previously described SDF-1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or “decoy” receptor has been in debate. It is known that CXCR7 is not expressed in normal blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed. The aim of this study was to determine the expression and function of macrophage CXCR7 linked to atherosclerosis. Here, we show that CXCR7 was detected in macrophage-positive area of aortic atheroma of ApoE-null mice, but not in healthy aorta. During monocyte differentiation to macrophages, CXCR7 was up-regulated at mRNA and protein levels, with more expression in M1 than in M2 phenotype. In addition, CXCR7 induction was associated with a SDF-1 signaling switch from the pro-survival ERK and AKT pathways in monocytes to the pro-inflammatory JNK and p38 pathways in macrophages. The latter effect was mimicked by a CXCR7-selective agonist TC14012 and abolished by siRNA knockdown of CXCR7. Furthermore, CXCR7 activation increased macrophage phagocytic activity, which was suppressed by CXCR7 siRNA silencing or by inhibiting either the JNK or p38 pathways, but was not affected by blocking CXCR4. Finally, activation of CXCR7 by I-TAC showed a similar signaling and phagocytic activity in macrophages with no detectable CXCR3. We conclude that CXCR7 is induced during monocyte-to-macrophage differentiation, which is required for SDF-1 and I-TAC signaling to JNK and p38 pathways, leading to enhanced macrophage phagocytosis, thus possibly contributing to atherogenesis.     

Selenium in serum and whole blood in patients with surgical interventions

Selenium values of serum and whole blood were determined for two groups of patients undergoing large surgical interventions (e.g., gastrectomy) and receiving a total parenteral nutrition (TPN). All patients showed low selenium values at the beginning of TPN. The first group received a supplementation of selenium, and it was established that selenium levels normalized after 6–7 d. The balances of selenium turned out to be positive in all cases. The second group, which was not administered selenium supplementation, showed significantly lower values of selenium. It can, therefore, be concluded that a substitution of selenium prior to large surgical interventions following TPN clearly helps patients combat the increase in the oxidant stress reactions.     

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.     

Signal Relay by CC Chemokine Receptor 2 (CCR2) and Formylpeptide Receptor 2 (Fpr2) in the Recruitment of Monocyte-derived Dendritic Cells in Allergic Airway Inflammation

Chemoattractant receptors regulate leukocyte accumulation at sites of inflammation. In allergic airway inflammation, although a chemokine receptor CCR2 was implicated in mediating monocyte-derived dendritic cell (DC) recruitment into the lung, we previously also discovered reduced accumulation of DCs in the inflamed lung in mice deficient in formylpeptide receptor Fpr2 (Fpr2^−/−). We therefore investigated the role of Fpr2 in the trafficking of monocyte-derived DCs in allergic airway inflammation in cooperation with CCR2. We report that in allergic airway inflammation, CCR2 mediated the recruitment of monocyte-derived DCs to the perivascular region, and Fpr2 was required for further migration of the cells into the bronchiolar area. We additionally found that the bronchoalveolar lavage liquid from mice with airway inflammation contained both the CCR2 ligand CCL2 and an Fpr2 agonist CRAMP. Furthermore, similar to Fpr2^−/− mice, in the inflamed airway of CRAMP^−/− mice, DC trafficking into the peribronchiolar areas was diminished. Our study demonstrates that the interaction of CCR2 and Fpr2 with their endogenous ligands sequentially mediates the trafficking of DCs within the inflamed lung.     

Comparative intracellular cytokine production by in vitro stimulated T lymphocytes from human umbilical cord blood (HUCB) and adult peripheral blood (APB).

To date over 400 HUCB transplants have been reported from different centers. It has been suggested that there is a reduced graft-versus-host-disease (GVHD) with HUCB compared to bone marrow transplantation. Since cytokine production by a cell is an indication of the cells function it is important to determinate the differences between APB and HUCB with respect to production of these soluble factors. Our aim was to analyse the intracellular cytokine production by HUCB and APB T lymphocytes with and emphasize on their possible role in GVHD. Heparinized HUCB samples from 8 normal full-term deliveries and 10 normal blood donors were stimulated 4 hours at 37 degrees C and 5% CO2 with phorbol 12-myristate 13-acetate (PMA) and lonomycin in the presence of brefeldine. Afterwards cells were stained with CD3, CD4 or CD8 in different combinations. Finally, after cell permeabilization, cells were stained with Il-2, Il-4 or IFN-gamma. Data acquisition was performed on a FACScan flow cytometer. Compared to APB, HUCB T lymphocytes produced less Il-2, Il-4 and IFN-gamma. In HUCB, Il-2, Il-4 and IFN-gamma were produced predominantly by CD4+ T cells. In APB, Il-2 and Il-4 were also produced predominantly by CD4+ cells compared with CD8+ T lymphocytes, however, IFN-gamma was produced by both CD4+ and CD8+ T cells. These results indicate that there are clear differences in the cytokine profile between T cells in APB and HUCB.     

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.