Antibacterial Activity on Orthopedic Clinical Isolates and Cytotoxicity of the Antimicrobial Peptide Dadapin-1.

In orthopedic surgery, biomaterial-associated infections represent a complication of serious concern. Most promising strategies to prevent these infections currently rely on the use of anti-infective biomaterials. Desirably, in anti-infective biomaterials, the antibacterial properties should be achieved by doping, grafting, or coating the material surfaces with molecules that are alternative to conventional antibiotics and exhibit a potent and highly specific activity against bacteria, without altering the biocompatibility. Antimicrobial peptides (AMPs) are among the most interesting candidate molecules for this biomaterial functionalization. Here, the potential expressed by the recently discovered peptide Dadapin-1 was explored by assaying its MIC, MBIC and MBC on clinical strains of relevant bacterial species isolated from orthopedic infections and by assessing its cytotoxicity on the human osteoblast-like MG63 cells. When appropriately tested in diluted Mueller Hinton Broth II (MHB II), Dadapin-1 exhibited significant antibacterial properties. MIC values were in the range of 3.1-6.2 µM for the gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri, and 12.4-24.9 µM for the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Interestingly, the peptide was found non-cytotoxic, with an IC50 exceeding the highest concentration tested of 179 µM. Overall, Dadapin-1 expresses considerable potential for future application in the production of anti-infective biomaterials.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

Synthesis, Crystal Structure, and Antibacterial Properties of Silver-Functionalized Low-Dimensional Layered Zirconium Phosphonates.

New insoluble layered zirconium phosphate carboxyaminophosphonates (ZPs), with the general formula Zr2(PO4)H5[(O3PCH2)2N(CH2)nCOO]2·mH2O (n = 3, 4, and 5), have been prepared and characterized. The crystal structure for n = 3 and 4 samples was determined ab initio from X-ray powder diffraction data. The structure for n = 3 was monoclinic in space group C2/c with the following unit cell parameters: a = 34.346(1) Å, b = 8.4930(2) Å, c = 9.0401(2) Å, and ß = 97.15(1)°. The structure for n = 4 was triclinic in space group P1̅ with the following unit cell parameters: a = 17.9803(9) Å, b = 8.6066(4) Å, c = 9.0478(3) Å, α = 90.466(3)°, ß = 94.910(4)°, and γ = 99.552(4)°. The two structures had the same connectivity as Zr phosphate glycine diphosphonate (n = 1), as previously reported. By intercalation of short amines, these layered compounds were exfoliated in single lamella or packets of a few lamellae, which formed colloidal dispersions in water. After a thorough characterization, the dispersed lamellae were functionalized with Ag nanoparticles, which were grown in situ on the surface of exfoliated lamellae. Finally, their antimicrobial activity was tested on several Gram-positive and Gram-negative bacteria. All of these systems were found to be active against the four pathogens most frequently isolated from orthopedic prosthetic infections and often causative of nosocomial infections. Interestingly, they were found to express powerful inhibitory activity even against bacterial strains exhibiting a relevant profile of antibiotic resistance such as Staphylococcus aureus ATCC 700699.

Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization.

Pollen grains induce allergies in susceptible individuals by release of allergens upon contact with mucosal membranes of the upper respiratory tract. We recently demonstrated that pollen not only function as allergen carriers but also as rich sources of bioactive lipids that attract cells involved in allergic inflammation such as neutrophils and eosinophils. Here we demonstrate that soluble factors from birch (Betula alba L.) pollen activate human dendritic cells (DCs) as documented by phenotypical and functional maturation and altered cytokine production. Betula alba L. aqueous pollen extracts (Bet.-APE) selectively inhibited interleukin (IL)-12 p70 production of lipopolysaccharide (LPS)- or CD40L-activated DC, whereas IL-6, IL-10, and TNFalpha remained unchanged. Presence of Bet.-APE during DC activation resulted in DC with increased T helper type 2 (Th2) cell and reduced Th1 cell polarizing capacity. Chemical analysis of Bet.-APE revealed the presence of phytoprostanes (dinor isoprostanes) with prostaglandin E(1)-, F(1)-, A(1)-, or B(1)-ring systems of which only E(1)-phytoprostanes dose dependently inhibited the LPS-induced IL-12 p70 release and augmented the Th2 cell polarizing capacity of DC. These results suggest that pollen-derived E(1)-phytoprostanes not only resemble endogenous prostaglandin E(2) structurally but also functionally in that they act as regulators that modulate human DC function in a fashion that favors Th2 cell polarization.

Pollen-derived E1-phytoprostanes signal via PPAR-gamma and NF-kappaB-dependent mechanisms.

In a humid milieu such as mucosal surfaces, pollen grains do not only release allergens but also proinflammatory and immunomodulatory lipids, termed pollen-associated lipid mediators. Among these, the E(1)-phytoprostanes (PPE(1)) were identified to modulate dendritic cell (DC) function: PPE(1) inhibit the DC's capacity to produce IL-12 and enhance DC mediated T(H)2 polarization of naive T cells. The mechanism(s) by which PPE(1) act on DC remained elusive. We thus analyzed candidate signaling elements and their role in PPE(1)-mediated regulation of DC function. Aqueous birch pollen extracts induced a marked cAMP response in DC that could be blocked partially by EP2 and EP4 antagonists. In contrast, PPE(1) hardly induced cAMP and the inhibitory effect on IL-12 production was mostly independent of EP2 and EP4. Instead, PPE(1) inhibited the LPS-induced production of IL-12 p70 by a mechanism involving the nuclear receptor PPAR-gamma. Finally, PPE(1) efficiently blocked NF-kappaB signaling in DCs by inhibiting IkappaB-alpha degradation, translocation of p65 to the nucleus, and binding to its target DNA elements. We conclude that pollen-derived PPE(1) modulate DC function via PPAR-gamma dependent pathways that lead to inhibition of NFkappaB activation and result in reduced DC IL-12 production and consecutive T(H)2 polarization.

Exploring the anticancer effects of standardized extracts of poplar-type propolis: In vitro cytotoxicity toward cancer and normal cell lines.

Propolis was shown to exert antimicrobial, antioxidant, anti-inflammatory, and anticancer activities. Its composition is influenced by seasonal, climatic and phytogeographic conditions. Further variability derives from the extraction methods. Multi Dynamic Extraction Method (MED) has been recently proposed to improve extracts reproducibility. Here, the cytotoxic/anticancer activity of three MED extracts of poplar-type propolis was assayed on human promyelocytic leukaemia HL60, human monocytic leukaemia THP-1, human osteosarcoma MG63, murine fibroblast L929 and human mesenchymal cells (hMSCs). As far as we are aware of, MG63 cells have never been challenged with propolis before, while few studies have so far addressed the effects of propolis on non-tumor cell lines. Consistent results were observed for all propolis preparations. The extracts turned out mildly cytotoxic toward cancer cells, in particular osteosarcoma cells (IC50: 81.9-86.7 µg/ml). Nonetheless, cytotoxicity was observed also in non-tumor L929 cells, with an even lower IC50. hMSCs demonstrated the lowest sensitivity to propolis (IC50: 258.3-287.2 µg/ml). In THP-1 cells, extracts were found to stimulate apoptosis caspase 3/7 activity. The IC50 values observed with osteosarcoma and leukaemia cells do not support a relevant cytotoxicity (as the figures abundantly exceeded 30 µg/ml), despites some selective activity exhibited with HL60 cells. The results confirm the validity of the extraction method, emphasizing the need to assess the selectivity of the interaction with cancer cells when screening for anticancer-drug candidates.

Pollen allergens do not come alone: pollen associated lipid mediators (PALMS) shift the human immune systems towards a T(H)2-dominated response.

Pollen allergy is characterized by a T(H)2-biased immune response to pollen-derived allergens. However, pollen-exposed epithelia do not encounter pure allergen but rather a plethora of protein and non-protein substances. We demonstrated that pollen liberate lipids with chemical and functional similarities to leukotriens and prostaglandins--the pollen associated lipid mediators (PALMs). To date, two main groups of PALMs have been characterized: The immunostimulatory PALMs activating innate immune cells such as neutrophils and eosinophils, and the immunomodulatory E(1)-phytoprostanes blocking IL-12 production of dendritic cells, resulting in the preferential induction of T(H)2 responses. This article reviews our work in the field of PALMs and their effects on cells of the innate and adoptive immune system. From recent results a general picture starts to emerge in which PALMs (and possibly other pollen-associated substances) may--independently from protein allergens--propagate an overall T(H)2 favoring micromilieu in pollen exposed tissue of predisposed individuals.

Antibody mimicry, receptors and clinical applications.

This review focuses on the concept of antibodies acting as receptor agonists and antagonists, and on the potential relevance of this notion in applied medicine. Antibodies are composed of three functional units: two antigen-binding fragments (Fabs) that confer antigen specificity and one constant fragment (Fc) linking antibodies to immune effector functions. The proof-of-concept that large amounts of highly specific and homogeneous antibodies could be produced was provided in 1975 by César Milstein and Georges Köhler. These monoclonal antibody (mAb) reagents started a revolution in medical research, diagnostics, and clinical applications. Alongside diagnostic applications, mAbs were successfully used in vivo: (i) to bind (neutralize/antagonize) antigens expressed on the surface of tumor cells; (ii) to activate immune effector mechanisms; (iii) to crosslink plasma membrane receptors and hence activate therapeutic signaling pathways; and lastly, (iv) the technique was expanded to produce bispecific mAbs, which can bind two different antigens while retaining the ability to activate immune effector functions. The abilities of mAbs to bind, transduce signals, and exert immunostimulatory agonistic capacities are the central issues of this review. The starting point is that some mAbs operate as molecular agonists, substituting for the natural ligand of the receptor. Our analysis is restricted to mAbs that act as receptor agonist/antagonists by either mimicking ligand binding, or through allosteric modulation mediated by binding sites that are topographically distinct from the orthosteric binding site. Functional considerations based on the agonistic stimulation of human CD38 by specific mAbs as surrogate ligands are described as examples of the features of such molecules.

Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles.

Higher efficacy and safety of nano gold therapeutics require examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells exposed to AuNP (5 and 30nm). Cellular internalization was dose and time-dependent for both AuNPs. The toxicity was observed by colony forming efficiency (CFE) and not by Trypan blue assay, and exclusively for 5nm AuNPs, starting at the concentration of 200µM (24 and 72h of exposure). The most pronounced changes in gene expression (Agilent microarrays) were detected at 72h (300µM) of exposure to AuNPs (5nm). The biological processes affected by smaller AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins were also differentially expressed. Our findings indicate that exposure to high concentration of AuNPs (5nm) induces metal exposure, oxidative stress signaling pathways, and might influence selenium homeostasis. Some of detected cellular responses might be explored as potential enhancers of anti-cancer properties of AuNPs based nanomedicines.

Activation of nickel-specific CD4+ T lymphocytes in the absence of professional antigen-presenting cells.

Allergic contact dermatitis ensues from exaggerated T cell responses to haptens. Dendritic cells are required for the initiation of hapten sensitization, but they may not be necessary for disease expression. Here we investigated the antigen-presenting cell requirement of nickel-specific CD4+ lymphocytes isolated from the blood of six allergic individuals. A significant proportion (42 out of 121; 35%) of the T cell clones proliferated in vitro to nickel also in the absence of professional antigen-presenting cells, suggesting a direct T-T hapten presentation. Antigen-presenting-cell-independent T cells showed a predominant T helper 1 phenotype. Nickel recognition by these T cells was major histocompatibility complex class II restricted, not influenced by CD28 triggering, independent from their state of activation, and did not require processing. The capacity of this T cell subset to be directly stimulated by nickel was not due to unique antigen-presenting properties, as both antigen-presenting-cell-dependent and antigen-presenting-cell-independent clones displayed comparable levels of HLA-DR, CD80, and CD86, and were equally capable of presenting nickel to antigen-presenting-cell-independent clones. In contrast, neither T cell types activated antigen-presenting-cell-dependent T lymphocytes. T-T presentation induced T cell receptor downregulation, CD25, CD80, CD86, and HLA-DR upregulation, and interferon-gamma release, although to a lesser extent compared to those induced by dendritic cell-T presentation. Following T-T presentation, the clones did not undergo unresponsiveness and maintained the capacity to respond to dendritic cells pulsed with antigen. In aggregate, our data suggest that antigen-presenting-cell-independent T cell activation can effectively amplify hapten- specific immune responses.

Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.

In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity.

Online monitoring of cell metabolism to assess the toxicity of nanoparticles: the case of cobalt ferrite.

Different in vitro assays are successfully used to determine the relative cytotoxicity of a broad range of compounds. Nevertheless, different research groups have pointed out the difficulty in using the same tests to assess the toxicity of nanoparticles (NPs). In this study, we evaluated the possible use of a microphysiometer, Bionas 2500 analyzing system Bionas GmbH®, to detect in real time changes in cell metabolisms linked to NPs exposure. We focused our work on response changes of fibroblast cultures linked to exposure by cobalt ferrite NPs and compared the results to conventional in vitro assays. The measurements with the microphysiometer showed a cobalt ferrite cytotoxic effect, confirmed by the Colony Forming Efficiency assay. In conclusion, this work demonstrated that the measurement of metabolic parameters with a microphysiometer is a promising method to assess the toxicity of NPs and offers the advantage to follow on-line the cell metabolic changes.

Glutathione peroxidase activity in the blood cells of psoriatic patients correlates with their responsiveness to Efalizumab.

Biological treatment of psoriasis, a chronic inflammatory immune-mediated pathology of huge social impact, has become a recent revolutionizing breakthrough in the management of the disease. Apart from anti-TNF-alpha biologics, recombinant proteins-inhibitors of the T lymphocytes-antigen presenting cells interaction, Efalizumab among them, have been successfully used in the therapy of psoriasis. Serious concern regarding safety and efficacy of biologics remains because they induce numerous adverse effects and a significant number of patients are non-responders. Up-to-now, there are no biochemical or/and immunological markers of the clinical efficacy of these drugs. This study searches for immunological and redox markers of the clinical response in the group of psoriatic patients treated with Efalizumab. Clinical response to Efalizumab was assessed by Psoriasis Area and Severity Index and correlated with suppression of T-cell functions, plasma cytokines, membrane-associated polyunsaturated fatty acids (PUFAs), antioxidant enzymes and markers of oxidative stress. A 12-week Efalizumab therapy did not affect abnormal plasma levels of pro-inflammatory cytokines and lower-than-normal content of PUFAs esterified in phospholipids of red cell membranes. It did, however, suppress T-cell-mediated functions and decrease nitrites/nitrates and malonyl dialdehyde levels independently on the clinical outcome. On contrast, activities of glutathione peroxidase (GPx) and glutathione S-transferase in granulocytes were remarkably increased and catalase decreased exclusively in non-responders vs complete or partial responders. High baseline GPx in erythrocytes decreased in responders. It is concluded that clinical response to Efalizumab correlates with GPx activity in the blood cells, suggesting that high hydroperoxide levels are involved in psoriasis persistence.

EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo.

Recent advances in the knowledge of the EGFR pathway have revealed its contribution to distinct immune/inflammatory functions of the epidermis. The purpose of our study was to evaluate the role of EGFR in the regulation of keratinocyte GM-CSF expression. In cultured human keratinocytes, proinflammatory cytokines synergized with TGF-alpha to induce GM-CSF expression. Accordingly, high epidermal levels of EGFR activation are associated with enhanced expression of GM-CSF in lesional skin of patients with psoriasis or allergic contact dermatitis. In cultured keratinocytes, pharmacological inhibition of EGFR activity reduced GM-CSF promoter transactivation, whereas genetic inhibition of AP-1 reduced expression of GM-CSF. Furthermore, EGFR activation enhanced TNF-alpha-induced c-Jun phosphorylation and DNA binding, whereas c-Jun silencing reduced GM-CSF expression. Using two different mouse models, we showed that the lack of a functional EGFR pathway was associated with reduced cytokine-induced phosphorylation of ERK1/2, JNK1/2, c-Jun and reduced keratinocyte-derived GM-CSF expression both in vitro and in vivo. Finally, the analysis of GM-CSF expression in the skin of cancer patients treated with anti EGFR drugs showed an association between ERK activity, c-Jun phosphorylation, and epidermal GM-CSF expression. These data demonstrate that the EGFR pathway is critical for the upregulation of keratinocyte GM-CSF expression under conditions of cytokine stimulation.

Gene expression study of two widely used pig intestinal epithelial cell lines: IPEC-J2 and IPI-2I.

The intestinal epithelial cells (IEC) play an important role in the immune system of swine, protecting against infectious and non-infectious environmental insults. The IEC participate in the innate immune response of the intestine through different mechanisms such as barrier function, mucus secretion, antibacterial peptide synthesis and participation in the cytokine/chemokine networks. Most of the current knowledge of intestinal cell functions has come from studies conducted on cell cultures generated from human cancers or from classical animal models. However, because the molecular and cellular elements of the immune system have been selected over evolutionary time in response to the species-specific environment, models of immune function based on mouse and human need to be applied cautiously in pig. Few models of swine small intestine epithelium exist and these are poorly characterised. In the present study we characterised the basal expression of epithelial and immune-related genes of two pig small intestine cell lines, IPEC-J2 and IPI-2I, under different culture conditions. These data represent essential background information for future studies on pig-intestinal pathogen interactions.

Plant polyphenols effectively protect HaCaT cells from ultraviolet C-triggered necrosis and suppress inflammatory chemokine expression.

Oxidative stress is a common response of epidermal cells to a variety of noxious stimuli such as ultraviolet (UV) radiation from solar light and proinflammatory cytokines from skin-infiltrating leukocytes. Here, we report that two types of plant-derived antioxidants, the phenylpropanoid glycoside verbascoside as well as the flavonoids rutin and quercetin possess protective effects against UVC-induced cell damage and proinflammatory activation. The molecules under investigation were effective against the loss of cell integrity associated with necrosis in doses consistent with their antioxidant activity, whereas they did not significantly oppose UVC-induced proliferation arrest and apoptosis. By contrast, only verbascoside effectively inhibited cytokine-induced release of proinflammatory mediators in a dose-dependent fashion. Verbascoside and its homologue teupolioside dramatically impaired NF-kappaB and AP-1 DNA binding activity. These results suggest that plant polyphenols with antioxidant properties have distinct mechanisms in the suppression of oxidative stress induced in keratinocytes by different stimuli. Verbascoside and teupolioside are hence of potential interest in the protection of the skin from both environmental and inflammatory insults.

The epidermal growth factor receptor system in skin repair and inflammation.

The epidermal growth factor (EGF) family comprises multiple mediators such as transforming growth factor-alpha, amphiregulin, heparin binding-EGF, and epiregulin, which are crucially involved in the tissue-specific proliferation/differentiation homeostasis. Typically, they act in an autocrine and paracrine manner on their specific cell membrane receptor and mount an effective reparative response to any attack to biophysical integrity. In addition, the EGFR can be activated by transactivation from a variety of G-protein-coupled receptors, integrins, and cytokine receptors, so that it acts as the major transducer of disparate cell functions, including changes in proliferation rate, cellular shape, attachment and motility, and regulation of proinflammatory activation. However, numerous experimental observations indicate that the different EGFR ligands are not redundant, but may rather provide distinct and specific contributions to keratinocyte functions. Importantly, increasing evidence now suggests that the EGFR pathway has a major impact on the inflammatory/immune reactions of the skin, in the apparent effort of enhancing innate immune defense while opposing overactivation of keratinocyte pro-inflammatory functions. This review covers the molecular mechanisms and functions activated by this major growth factor system in the regulation of keratinocyte biology and focuses on the complex contribution of EGFR signaling to the inflammatory processes in the skin.

A comparative gene index for the white sturgeon Acipenser transmontanus.

Sturgeons are archaic fishes phylogenetically distinct from Teleosts. They represent an important niche for aquaculture, particularly for the production of caviar and high quality fillets, while many natural populations in various world areas are today threatened by extinction. Knowledge of the sturgeon genome is limited, as it is the case of many other species of interest for fishery, aquaculture and conservation. Sequences from non-normalized libraries of skin and spleen of the American sturgeon (A. transmontanus) produced in our laboratories were analysed via a bioinformatic procedure, and compared to similar resources available for three Teleost species. Data collected during the analyses were stored in a database - the Sturgeon database (db) - that can be queried via a web interface. The Sturgeon db contains a total of 16,404 sequences from Acipenser transmontanus, Ictalurus punctatus, Salmo salar and Takifugu rubripes, each specie being represented by expressed sequence tags (ESTs) from skin and spleen. Data contained in the database are the results of a number of analyses that mostly focus on sequence annotation and intra- and inter-species comparison. Putative SNP sites, tandem repeats, and sequences matching known protein patterns and motifs were also identified. The Sturgeon db is by now the only online resource dedicated to the analysis of A. transmontanus EST sequences, and represents a starting point for the investigation of the genome of sturgeons from a physiological perspective; it will be used to identify polymorphic markers to study, for example, fish pathologies or to survey fish disease resistance, and to produce gene expression arrays. Introduction of sequences from other species in the analysis pipeline allowed inter-species comparisons of transcripts distribution in Gene Ontology categories, as well as orthologs identification, despite the high sturgeon phylogenetic distance from other fish species. As a result of the EST analysis procedure, 1058 sturgeon novel unigenes were identified.

Immunomodulatory mediators from pollen enhance the migratory capacity of dendritic cells and license them for Th2 attraction.

The immune response of atopic individuals against allergens is characterized by increased levels of Th2 cytokines and chemokines. However, the way in which the cytokine/chemokine profile is matched to the type of invading allergen, and why these profiles sometimes derail and lead to disease, is not well understood. We recently demonstrated that pollen modulates dendritic cell (DC) function in a way that results in an enhanced capacity to initiate Th2 responses in vitro. Here, we examined the effects of aqueous birch pollen extracts (Bet.-APE) on chemokine receptor expression and chemokine production by human monocyte-derived DCs. Bet.-APE strongly induced expression and function of CXCR4 and reduced CCR1 and CCR5 expression on immature DCs. In addition, DC treatment with Bet.-APE significantly reduced LPS-induced production of CXCL10/IP-10, CCL5/RANTES; induced CCL22/macrophage-derived chemokine; and did not significantly change release of CCL17/thymus and activation-regulated chemokine. At a functional level, Bet.-APE increased the capacity of LPS-stimulated DCs to attract Th2 cells, whereas the capacity to recruit Th1 cells was reduced. Bet.-APE significantly and dose-dependently enhanced intracellular cAMP, suggesting that water-soluble factors from pollen grains bind a G(alphas)-protein-coupled receptor. E(1)-Phytoprostanes were identified to be one player in the Th2-polarizing potential of aqueous pollen extracts. In summary, our results demonstrate that pollen itself releases regulatory mediators which generate a Th2-promoting micromilieu with preferential recruitment of Th2 cells to the site of pollen exposure.