Deficiency of leukocyte-specific protein 1 (LSP1) alleviates asthmatic inflammation in a mouse model.

BACKGROUND: Asthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation. However, its role in asthma remains unknown. METHODS: An OVA-induced mouse asthma model in LSP1-deficient (Lsp1-/-) and wild-type (WT) 129/SvJ mice were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung inflammation. RESULTS: Light and electron microscopic immunocytochemistry and Western blotting showed that, compared with normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to Lsp1-/- OVA mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05). The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1-/- OVA mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in Lsp1-/- OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of Lsp1-/- OVA mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially associated with the vascular endothelium and neutrophils. CONCLUSION: These data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation, including leukocyte recruitment and cytokine expression, in a mouse model of asthma.

Therapeutic reversal of food allergen sensitivity by mature retinoic acid-differentiated dendritic cell induction of LAG3+CD49b-Foxp3- regulatory T cells.

BACKGROUND: Anaphylaxis is a life-threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients. OBJECTIVE: We assessed whether DC immunotherapy could reverse food allergen sensitivity in mouse models to provide proof of concept relating to their use in the clinic. METHODS: We generated and characterized mature retinoic acid-skewed dendritic cells (DC-RAs) and assessed their abilities to reverse ovalbumin or peanut allergies in mouse models, as well as their operative mechanisms. RESULTS: DC-RAs displayed a mature yet tolerogenic phenotype, expressing IL-10, TGF-ß, IL-27, and aldehyde dehydrogenase 1A2 but not IL-12 or IL-35; IL-10 and TGF-ß together drove their suppression of TH2 cell proliferation. Delivery of specific allergen-presenting DC-RAs to half-maximally sensitized mice with ovalbumin or peanut allergy reduced anaphylactic responses to oral allergen challenge by 84% to 90%, as well as diarrhea, mast cell activation, and TH2 cytokine responses and serum allergen-specific IgE/IgG1 levels. DC-RA expression of IL-27 was important to their induction of CD25+ lymphocyte activation gene 3 (LAG3)+, CD49b-, forkhead box P3 (Foxp3)- regulatory T cells in vitro, such that ß subunit of IL-27 (Ebi)-/- (ie, IL-27-incompetent) DC-RAs were ineffective in inducing food allergen tolerance. CONCLUSION: Our data indicate that regulatory DC immunotherapy can be effective for food allergies and suggest that induction of Foxp3- regulatory T cells might be a useful strategy for tolerance induction in this context.

Effects of K11R and G31P Mutations on the Structure and Biological Activities of CXCL8: Solution Structure of Human CXCL8(3-72)K11R/G31P.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), the expression levels of which are elevated in many inflammatory diseases, binds to both the CXCR1 and CXCR2 receptors with high affinity. Recently, an analogue of human CXCL8, CXCL8(3-72)K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both the CXCR1 and CXCR2. Herein, we have determined the solution structure and the CXCR1 N-terminal peptide binding sites of hG31P by NMR spectroscopy. We have found that the displacement within the tertiary structure of the 30 s loop and the N-terminal region and more specifically change of the loop conformation (especially H33), of hG31P may affect its binding to the CXCR1 receptor and thereby inhibit human neutrophil chemotactic responses induced by ELR-CXC chemokines. Our results provide a structural basis for future clinical investigations of this CXCR1/CXCR2 receptor antagonist and for the further development of CXCL8 based antagonists.

Comparison of induced versus natural regulatory T cells of the same TCR specificity for induction of tolerance to an environmental antigen.

Recent evidence shows that natural CD25(+)Foxp3(+) regulatory T cells (nTreg) and induced CD25(+)Foxp3(+) regulatory T cells (iTreg) both contribute to tolerance in mouse models of colitis and asthma, but there is little evidence regarding their relative contributions to this tolerance. We compared the abilities of nTreg and iTreg, both from OVA-TCR-transgenic OTII mice, to mediate tolerance in OVA-asthmatic C57BL/6 mice. The iTreg were differentiated from Th2 effector T cells by exposure to IL-10-differentiated dendritic cells (DC10) in vitro or in vivo, whereas we purified nTreg from allergen-naive mice and exposed them to DC10 before use. Each Treg population was subsequently repurified and tested for its therapeutic efficacy in vitro and in vivo. DC10 engaged the nTreg in a cognate fashion in Forster (or fluorescence) resonance energy transfer assays, and these nTreg reduced in vitro OVA-asthmatic Th2 effector T cell responses by 41-56%, whereas the comparator iTreg reduced these responses by 72-86%. Neutralization of IL-10, but not TGF-ß, eliminated the suppressive activities of iTreg but not nTreg. Delivery of 5 × 10(5) purified nTreg reduced allergen challenge-induced airway IL-4 (p ≤ 0.03) and IL-5 (p ≤ 0.001) responses of asthmatic recipients by ≤ 23% but did not affect airway hyperresponsiveness or IgE levels, whereas equal numbers of iTreg of identical TCR specificity reduced all airway responses to allergen challenge by 82-96% (p ≤ 0.001) and fully normalized airway hyperresponsiveness. These data confirm that allergen-specific iTreg and nTreg have active roles in asthma tolerance and that iTreg are substantially more tolerogenic in this setting.

Induction of prolonged asthma tolerance by IL-10-differentiated dendritic cells: differential impact on airway hyperresponsiveness and the Th2 immunoinflammatory response.

IL-10-differentiated dendritic cells (DC10s) can prevent allergen sensitization and reverse the asthma phenotype in mice with established disease. However, little is known about the time-frames over which this tolerance is effective. We report that at 2 wk after i.p. or transtracheal delivery of 1 × 10(6) OVA-, but not house dust mite- presenting, DC10s to OVA-asthmatic mice, significant diminution of airway hyperresponsiveness (AHR) was first apparent, whereas AHR was abrogated between 3 and 10 wk posttreatment. At 13 wk, AHR returned to pretreatment levels but could again be reversed by DC10 retreatment. The impact of a single DC10 treatment on airway eosinophil and Th2 cytokine responses to recall OVA challenge, and on OVA-specific IgE/IgG1 responses, was substantial at 3 wk posttreatment, but progressively increased thereafter, such that at 8 mo, airway eosinophil and Th2 responses to recall allergen challenge remained ∼85-95% suppressed relative to saline-treated asthmatic mice. Four biweekly DC10 treatments, whether transtracheal or i.p., reduced all asthma parameters to near background by 8 wk, whereas s.c. DC10 treatments did not affect AHR but did reduce the airway Th2 responses (i.v. DC10 had no discernible effects). Repeated challenge of the DC10-treated mice with aerosolized OVA (100 µg/ml) did not reverse tolerance, but treatment with the indoleamine-2,3-dioxygenase antagonist 1-methyltryptophan or neutralizing anti-IL-10R from days 12 to 21 after DC10 therapy partially reversed tolerance (Th2 cytokine responses, but not AHR). These findings indicate that DC10-induced Th2 tolerance in asthmatic animals is long lived, but that DC10s employ distinct mechanisms to affect AHR versus Th2 immunoinflammatory parameters.

Recombinant human CXCL8(3-72)K11R/G31P regulates smooth muscle cell proliferation and migration through blockage of interleukin-8 receptor.

Atherosclerosis is a chronic inflammatory disease with multiple contributing factors. Hyperlipidemia is one of the major independent risks, and interleukin-8 (IL-8), as an inflammatory factor, plays an important role in the development of atherosclerosis. The aims of the study were to examine the therapeutic efficacy of G31P, an antagonist of IL-8 receptor, with a mouse model of hyperlipidemia and the potential mechanisms of G31P through the vascular smooth muscle cell (VSMC) proliferation and migration in a cell line. In vivo study: Male BALB/c mice were fed a high-fat diet for 6 months. G31P was injected subcutaneously. Blood keratinocyte chemoattractant, lipid profile, and aorta expression of inflammatory factors, matrix metalloproteinases, MMP2 and MMP9 were investigated. In vitro study: A7R5 cells were treated with IL-8 with/without G31P. Cell proliferation and migration were investigated. G31P significantly suppressed the hyperlipidermia-induced abnormal lipid profile and increased IL-8, proinflammatory factor, MMP2 and MMP9 expression. G31P also inhibited VSMC proliferation and migration both in vitro and in vivo. These findings indicate the potential therapeutic effects of G31P in preventing the development of atherosclerosis by antagonizing IL-8 receptor and decreasing the biologic activity of IL-8.

Mucosal allergic sensitization to cockroach allergens is dependent on proteinase activity and proteinase-activated receptor-2 activation.

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.

Tolerogenic dendritic cells induce CD4+CD25hiFoxp3+ regulatory T cell differentiation from CD4+CD25-/loFoxp3- effector T cells.

IL-10-differentiated dendritic cells (DC10) induce allergen tolerance in asthmatic mice, during which their lung Th2 effector T cells (Teffs) are displaced by activated CD4(+)CD25(hi)Foxp3(+) T cells. Intestinal DCs promote oral tolerance by inducing Ag-naive T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), but whether DCs can induce Teffs to differentiate into Tregs remains uncertain. In this study, we addressed this question in OVA-asthmatic mice that were treated with DC10. OVA-presenting DC10 treatment maximally activated lung Tregs in these animals at 3 wk posttreatment, as determined by upregulation of activation markers (ICOS, programmed cell death-1, glucocorticoid-induced TNFR-related protein, LAG3, and CTLA-4) and in functional assays. This in vitro regulatory activity was ≥90% reduced by treatment with anti-IL-10 but not anti-TGF-ß Abs. In parallel cultures, OVA- but not house dust mite (HDM)-presenting DC10 induced ≈43% of CFSE-labeled CD25(-/lo)Foxp3(-) Teffs from asthmatic OVA-TCR transgenic mice to differentiate into tolerogenic CD25(hi)Foxp3(+) Tregs. We recapitulated this in vivo using OVA-asthmatic mice that were coinjected with OVA- or HDM-presenting DC10 (i.p.) and CFSE-labeled CD4(+)CD25(-/lo)Foxp3(-) Teffs (i.v.) from the lungs of asthmatic DO11.10 mice. From ≈7 to 21% of the activated (i.e., dividing) DO11.10 Teffs that were recovered from the lungs, lung-draining lymph nodes, or spleens of the OVA-DC10 recipients had differentiated into CD4(+)CD25(hi)Foxp3(+) Tregs, whereas no CFSE-positive Tregs were recovered from the HDM-DC10-treated animals. These data indicate that DC10 treatments induce tolerance at least in part by inducing Teffs to differentiate into CD4(+)CD25(hi)Foxp3(+) Tregs.

G31P, an antagonist against CXC chemokine receptors 1 and 2, inhibits growth of human prostate cancer cells in nude mice.

Prostate cancer is the most common malignancy in Western countries. Chemokine C-X-C motif receptor 1 (CXCR1) and CXCR2 play a key role in generation and regulation of CXC chemokine signaling. CXCR1 is a receptor for interleukin 8 (IL8), a pro-inflammatory chemokine, and CXCR1/2 are crucially involved in the prostate cancer development and progression. Thus, we generated a high-affinity human CXCR1/CXCR2 inhibitor, CXCL8 (3-72) K11R/G31P, named G31P, which is a synthetic derivative of the human cytokine, IL-8. In this study, we investigated the effects of G31P on regulation of prostate cancer cell growth in vitro and in nude mouse xenografts. Cell viability, adhesion, and wound healing assays were used to assess the effects of G31P on growth, adhesion, and migration of PC-3 human prostate cancer cells in vitro, respectively. Nude mouse xenografts and xenograft implantation assays were performed to determine the effect of G31P on PC-3 cells in vivo. Immunohistochemistry was used to detect gene expression, and fluorescence imaging was used to detect tumor volume and microvessel density in tumor xenografts. The data showed that G31P treatment significantly reduced PC-3 cell viability, adhesion and migration capacity in a dose-dependent manner (up to 100 ng/ml). Additionally, G31P treatment of nude mice suppressed the growth of orthotopically transplanted tumor xenografts. G31P also inhibited tumor tissue vascularization, which was associated with the decreased expression of vascular endothelial growth factor and nuclear transcription factor (NF)-κB in orthotopic xenograft tissues. This study provides evidence that G31P, a CXCR1/2 inhibitor, may effectively control prostate cancer.

ELR-CXC chemokine receptor antagonism targets inflammatory responses at multiple levels.

The ELR-CXC chemokines play important roles in neutrophilic inflammation. We report in this study that a fully human ELR-CXC chemokine antagonist that we have generated, CXCL8((3-72))K11R/G31P (G31P), has potent anti-inflammatory effects that arise through its actions at multiple levels. G31P inhibited CXCL8-induced chemotactic responses and intracellular Ca(2+) flux in CXCR1-transfected HEK cells and neutrophils, and responses of neutrophils to CXCR2-exclusive ligands. G31P desensitized heterologous G protein-coupled receptors on neutrophils, 52-86% reducing their Ca(2+) flux and chemotactic responses to leukotriene B(4), C5a, and the bacterial tripeptide fMLP. G31P also 60-90% blocked neutrophil chemotactic responses to mediators present in 10 of 12 sputum samples from cystic fibrosis or bronchiectasis subjects with bacterial pneumonia. Moreover, whereas A549 bronchial epithelial cells (which expressed CXCR1) secreted approximately 29,000 pg/ml CXCL8 in response to in vitro endotoxin challenge, G31P reduced this response by up to 98%, presumably by interrupting an autocrine inflammatory loop. The anti-inflammatory effects of G31P extended also to reversing the antiapoptotic influence of ELR-CXC chemokines on neutrophils. That these effects were relevant in vivo was confirmed in a guinea pig model of airway endotoxemia, wherein the human form of G31P >95% blocked neutrophil infiltration into and activation within the airways, as determined by airway levels of the neutrophil primary, secondary, and tertiary granule markers myeloperoxidase, lactoferrin, and matrix metalloproteinase-9, respectively, and the epithelial cell marker matrix metalloproteinase-2. These data suggest that the beneficial effects of ELR-CXC chemokine antagonism arise through effects that occur at multiple levels, including epithelial cells, neutrophils, and alternate G protein-coupled receptors.

Regulatory Dendritic Cells, T Cell Tolerance, and Dendritic Cell Therapy for Immunologic Disease.

Dendritic cells (DC) are antigen-presenting cells that can communicate with T cells both directly and indirectly, regulating our adaptive immune responses against environmental and self-antigens. Under some microenvironmental conditions DC develop into anti-inflammatory cells which can induce immunologic tolerance. A substantial body of literature has confirmed that in such settings regulatory DC (DCreg) induce T cell tolerance by suppression of effector T cells as well as by induction of regulatory T cells (Treg). Many in vitro studies have been undertaken with human DCreg which, as a surrogate marker of antigen-specific tolerogenic potential, only poorly activate allogeneic T cell responses. Fewer studies have addressed the abilities of, or mechanisms by which these human DCreg suppress autologous effector T cell responses and induce infectious tolerance-promoting Treg responses. Moreover, the agents and properties that render DC as tolerogenic are many and varied, as are the cells' relative regulatory activities and mechanisms of action. Herein we review the most current human and, where gaps exist, murine DCreg literature that addresses the cellular and molecular biology of these cells. We also address the clinical relevance of human DCreg, highlighting the outcomes of pre-clinical mouse and non-human primate studies and early phase clinical trials that have been undertaken, as well as the impact of innate immune receptors and symbiotic microbial signaling on the immunobiology of DCreg.

CD40 signaling augments IL-10 expression and the tolerogenicity of IL-10-induced regulatory dendritic cells.

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells' abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.

Atopy risk among school-aged children in relation to early exposures to a farm environment: A systematic review.

BACKGROUND AND OBJECTIVES: Childhood atopy is a complex condition with both a genetic and an environmental component. This systematic review will explore the current understanding of the importance of early life exposures to a farm in the development of atopy measured by objective markers of skin prick testing, and specific IgE measurements in school age children. METHODS: A systematic review was performed. RESULTS: Among 7285 references identified, 14 studies met the inclusion criteria (13 cross-sectional studies and 1 case-control study). The results were fairly consistent in that early farm-related exposures can protect children from becoming atopic at school age. In general, there was heterogeneity in the assessment of outcomes and exposures. CONCLUSIONS: Early-life farm exposures are associated with a protective effect on childhood atopy as assessed by objective markers. Future work should focus on understanding specific farm exposures that may important in these associations between atopy and farm exposures in children.

Soluble Low-density Lipoprotein Receptor-related Protein 1 in Juvenile Idiopathic Arthritis.

OBJECTIVES: This study aimed to expand knowledge about soluble low-density lipoprotein receptor-related protein 1 (sLRP1) in juvenile idiopathic arthritis (JIA) by determining associations of sLRP1 levels in nonsystemic JIA patients with clinical and inflammatory biomarker indicators of disease activity. METHODS: Plasma sLRP1 and 44 inflammation-related biomarkers were measured at enrollment and 6 months later in a cohort of 96 newly diagnosed Canadian patients with nonsystemic JIA. Relationships between sLRP1 levels and indicators of disease activity and biomarker levels were analyzed at both visits. RESULTS: At enrollment, sLRP1 levels correlated negatively with age and active joint counts. Children showed significantly higher levels of sLRP1 than adolescents (mean ranks: 55.4 and 41.9, respectively; P = 0.02). Participants with 4 or fewer active joints, compared to those with 5 or more active joints, had significantly higher sLRP1 levels (mean ranks: 56.2 and 40.7, respectively; P = 0.006). At enrollment, considering the entire cohort, sLRP1 correlated negatively with the number of active joints (r = -0.235, P = 0.017). In the entire cohort, sLRP1 levels at enrollment and 6 months later correlated with 13 and 6 pro- and antiinflammatory biomarkers, respectively. In JIA categories, sLRP1 correlations with inflammatory markers were significant in rheumatoid factor-negative polyarticular JIA, oligoarticular JIA, enthesitis-related arthritis, and psoriatic arthritis at enrollment. Higher sLRP1 levels at enrollment increased the likelihood of absence of active joints 6 months later. CONCLUSION: Plasma sLRP1 levels correlate with clinical and biomarker indicators of short-term improvement in JIA disease activity, supporting sLRP1 as an upstream biomarker of potential utility for assessing JIA disease activity and outcome prediction.

Induction of type 2 T helper cell allergen tolerance by IL-10-differentiated regulatory dendritic cells.

In mouse models of asthma, therapeutic use of allergen-presenting IL-10-differentiated dendritic cells (DCs) can abrogate airway hyperresponsiveness, and reduce other asthma-related responses to near background. Analogous human DCs can suppress human T cell responses in vitro, but the operative mechanisms are poorly defined. We investigated the ability of IL-10-treated human DCs to induce tolerance among autologous T cells of subjects with asthma and the mechanisms by which they do this. CD14(+) monocyte-derived DCs were differentiated in the presence of IL-10 (DC10) ex vivo from 11 donors with asthma and 4 control donors, and characterized for relevant markers. They were pulsed with specific or irrelevant allergen, and cultured with autologous peripheral blood CD4(+) T cells, either alone or together with autologous immunostimulatory DCs (DC-TNF), and the impact of this treatment on the T-cell responses was assessed for each donor. The DC10 expressed reduced levels of some relevant markers (CD40, CD80, human leukocyte antigen-DR) and stimulatory cytokines (IL-6 and IL-12), but augmented levels of Ig-like transcript-22/CD85j and IL-10 relative to DC-TNF. In cocultures, they dampened DC-TNF-driven T helper (Th) type 2 cell proliferation and cytokine (IL-4, -5, and -13) secretion. They also drove the development from atopic CD4(+)CD25(lo)Foxp3(lo) cells of a population of IL-10-secreting CD25(+)Foxp3(+)LAG-3(+)CTLA-4(+) regulatory T cells (Tregs). These Tregs suppressed stimulatory DC-induced autologous Th2 cell proliferation and cytokine secretion in a contact-dependent manner. Our data indicate that IL-10-treated human DCs induce Th2 cell allergen tolerance ex vivo by driving the differentiation of Tregs.

Blockade of neutrophil responses in aspiration pneumonia via ELR-CXC chemokine antagonism does not predispose to airway bacterial outgrowth.

Pneumonia associated with aspiration of bacterial-laden gastric contents is characterized by Glu-Leu-Arg (ELR)-CXC chemokine (e.g., CXC2L1, CXCL8) expression leading to local neutrophil sequestration. This neutrophil response is designed to be protective, but overly aggressive responses can be pathogenic in themselves. Herein we assessed whether blocking neutrophil responses in a guinea pig model of aspiration pneumonia would foster airway bacterial growth. Guinea pigs (n=5) were challenged intranasally with saline, acidified saline or acidified gastric contents (35mg/kg body weight, pH 2.0) and treated subcutaneously with 250mug/kg of the human ELR-CXC chemokine antagonist CXCL8((3-72))K11R/G31P (G31P) or saline. After 20h the animals' airway inflammatory responses and bacterial burdens were assessed. A loss of vascular integrity was apparent in the lungs of the saline-treated aspiration pneumonia animals (12.07+/-1.3% of the pleural surfaces exhibited hemorrhagic consolidation, 4.6x10(6) RBC/ml bronchoalveolar lavage fluid [BALF]), as was a pulmonary neutrophilia. The BAL fluids contained gram-negative and -positive bacteria (total load, 6.3+/-3.2x10(7) CFU/ml BALF) that are associated with nosocomial infections in humans. The G31P-treatments attenuated the pulmonary vascular complications (2.27+/-0.5% pleural surface hemorrhagic consolidation, 0.46x10(6) RBC/ml BALF), and reduced the pulmonary neutrophilia by >/=86%. The G31P-treatments did not lead to significant changes in the airway bacterial loads (total load, 3.46+/-1.8x10(7) CFU/ml BALF). This data indicates that attenuation of the pulmonary neutrophil response in aspiration pneumonia reduces pathology very significantly but does not reduce the efficiency of pulmonary bacterial clearance.

A novel ELR-CXC chemokine antagonist reduces intestinal ischemia reperfusion-induced mortality, and local and remote organ injury.

BACKGROUND: Neutrophil sequestration plays an important role in mediating local and remote organ injury induced by ischemia and reperfusion (I/R). The Glu-Leu-Arg (ELR)-CXC subfamily of chemokines, all CXCR1 or CXCR2 ligands, are primary agonists for such neutrophil recruitment. Herein, we assessed the effects of a combined CXCR1/CXCR2 antagonist, CXCL8((3-72))K11R/G31P (G31P), on neutrophilic local (gut) and distant organ injury and outcomes after superior mesenteric artery I/R in rats. METHODS: Male Sprague-Dawley rats (n=6-10) were subjected to either sham treatment or superior mesenteric artery ischemia for 1h; all animals received either saline or G31P (500 mug/kg, s.c.) and were euthanized for assessment after either 2 or 5h of arterial reperfusion. Survival and gut pathology, and pulmonary neutrophils were assessed directly, while bronchoalveolar lavage (BAL) fluid total protein levels and red blood cell (RBC) numbers were determined by protein assay and direct counting. Expression of inflammatory mediators in the lung and jejunum was measured by quantitative RT-PCR, colorimetric or gel zymography assays. RESULTS: Sham treatment animals suffered no discernible gut or pulmonary pathology. At 2 and 5h after reperfusion, the survival levels of the saline-treated I/R injury animals were 80% and 50%, respectively, while all G31P-treated animals survived. I/R injury led to substantial villous pathology within the jejunum, and G31P significantly reduced these pathology scores as well as neutrophil infiltration of the jejunal lamina propria and lung parenchyma, and vascular leakage into the airways (BAL protein). The tissue injury increased expression of myeloperoxidase and matrix metalloproteinase (MMP)-2 and MMP-9 in the gut tissues, but G31P treatment did not significantly affect this response. Intestinal I/R increased expression of IL-1, IL-6, GRO, and MIP-2 in the ischemic jejunum and the lung tissues, but here too G31P treatment had no palliative effects on these responses. CONCLUSION: These results suggest that full-spectrum ELR-CXC chemokine antagonism has significant protective effects against I/R-induced local and remote organ injury.

The effect of early-life stress on airway inflammation in adult mice.

BACKGROUND/AIMS: Neonatal stress induces permanent physiological changes that may influence the immune system. Early-life stress increases asthma disease severity in children. We investigated the effects of early-life stress on allergic airway inflammation using a murine model of asthma coupled to maternal separation as an early-life stress stimulus. METHODS: Maternally separated (MS) and unseparated control (CON) mice were sensitized with ovalbumin (OVA) beginning at day 31 after birth. RESULTS: Challenging mice with OVA increased airway hyperresponsiveness (AHR) and the number of inflammatory cells recovered in the bronchoalveolar lavage (BAL), compared to saline-challenged mice. Challenging MS mice with OVA resulted in less total inflammatory cells, eosinophils, interferon-gamma, and interleukin-4 in BAL compared to CON mice. However, MS mice challenged with OVA exhibited AHR similar to CON mice challenged with OVA. In contrast, an enhanced stress protocol (MS+) involving removal of pups from their home cages following the removal of the dam resulted in inflammatory cell accumulation and cytokine levels in the BAL similar to CON mice and higher than MS mice. CONCLUSIONS: These findings indicate that the effect of early-life psychological factors on the development of airway inflammatory diseases such as asthma is very complex and depends on the quality of the psychological stress stimulus.

Fractionation of swine barn dust and assessment of its impact on the respiratory tract following repeated airway exposure.

The effects of repeated exposure to a range of doses of swine barn dust (SBD) on airway hyperresponsiveness (AHR) and inflammation were evaluated using a mouse model system. A number of components, including endotoxin and a number of feed proteins, were identified in SBD, and mice were exposed 20 min/d for 14 d to a log dilution series of nebulized SBD suspensions. AHR to methacholine was measured using head-out whole-body plethysmography, and the methacholine concentration inducing a 20% decrease in pulmonary airflow (PC(20) MCh) was calculated. At the end of the 14-d exposure period, bronchoalveolar lavage (BAL) fluids were recovered, cytokines (interleukin [IL]-1beta, IL-6, keratinocyte-derived chemokine [KC], and tumor necrosis factor [TNF]) in BAL were measured by enzyme-linked immunosorbent assay (ELISA), and leukocytes in BAL were counted. The PC(20) MCh was significantly lower in the group of mice that were exposed to the highest concentration of SBD than in controls or the group exposed to the lowest level of dust. Likewise, the group that was exposed to the highest level of SBD had significantly higher levels of IL-1beta, KC, and TNF than controls and some other groups. There were substantially more lymphocytes and monocytes in the BAL from mice that were exposed to the higher levels of SBD for the 14-d period, but neutrophils were not a part of this response. The SBD exposures used in these experiments induced chronic inflammatory phenotype responses, as indicated by the predominance of lymphocytes and monocytes, but not neutrophils, in BAL and by inflammatory cytokines detected. The association between the PC(20)MCh and dose of SBD suggests that a threshold of susceptibility occurs after a relatively low, chronic exposure to SBD.

RadBall Technology Testing in the Savannah River Site's Health Physics Instrument Calibration Laboratory.

The United Kingdom's National Nuclear Laboratory (NNL) has developed a radiation-mapping device that can locate and quantify radioactive hazards within contaminated areas of the nuclear industry. The device, known as RadBall(™), consists of a colander-like outer collimator that houses a radiation-sensitive polymer sphere. The collimator has over two hundred small holes; thus, specific areas of the polymer sphere are exposed to radiation becoming increasingly more opaque in proportion to the absorbed dose. The polymer sphere is imaged in an optical-CT scanner that produces a high resolution 3D map of optical attenuation coefficients. Subsequent analysis of the optical attenuation data provides information on the spatial distribution of sources in a given area forming a 3D characterization of the area of interest. The RadBall(™) technology has been deployed in a number of technology trials in nuclear waste reprocessing plants at Sellafield in the United Kingdom and facilities of the Savannah River National Laboratory (SRNL). This paper summarizes the tests completed at SRNL Health Physics Instrument Calibration Laboratory (HPICL).