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Aims: To compare the vitreous levels of chemokines in diabetic patients with and without retinopathy. To find the relationship between stages of diabetic retinopathy (DR) and levels of vitreous chemokines., Methods: The study involved 20 non-diabetic and 20 diabetic patients without clinical signs of DR (NDR) and 40 diabetic patients with proliferative diabetic retinopathy (PDR). The vitreous humor was collected and the levels of 40 chemokines were measured using magnetic color-bead-based multiplex assay., Results: The control group, NDR group, PDR with vitreous hemorrhage (VH) group, and PDR with tractional retinal detachment group comprised 20, 20, 21, and 19 eyes, respectively. Only the concentration of CCL3 was significantly higher in the NDR group compared with the controls (p = 0.038). Twenty-five types of chemokines were statistically higher in the PDR with VH group in comparison to NDR group (all p < 0.05). All chemokines were statistically higher in the PDR with TRD group in comparison to NDR group (all p < 0.05) apart from 3 chemokines: GM-CSF, MIF, and CCL3(p = 0.086, p = 0.109, p = 0.094, respectively). The concentration of CCL21, CCL15 in PDR with TRD group was significantly higher compared with PDR with VH group, while other 36 chemokines were not significantly different between PDR with VH group and PDR with TRD group., Conclusions: The inflammation gradually worsen with the progression of DR. CCL3 may be associated with the onset of early diabetic retinal damage, and CCL15 and CCL21 may be closely related to the formation of fibrovascular membrane and the progression of the end stage of DR.

Severe acute pancreatitis is a lethal inflammatory disease frequently accompanied by pancreatic necrosis. We aimed to identify a key regulator in the development of pancreatic necrosis. A cytokine/chemokine array using sera from patients with acute pancreatitis (AP) revealed that serum CXCL16 levels were elevated according to the severity of pancreatitis. In a mouse model of AP, Cxcl16 expression was induced in pancreatic acini in the late phase with the development of pancreatic necrosis. Cxcl16 -/- mice revealed similar sensitivity as wild-type (WT) mice to the onset of pancreatitis, but better resisted development of acinar cell necrosis with attenuated neutrophil infiltration. A cytokine array and immunohistochemistry revealed lower expression of Ccl9, a neutrophil chemoattractant, in the pancreatic acini of Cxcl16 -/- mice than WT mice. Ccl9 mRNA expression was induced by stimulation with Cxcl16 protein in pancreatic acinar cells in vitro, suggesting a Cxcl16/Ccl9 cascade. Neutralizing antibody against Cxcl16 ameliorated pancreatic injury in the mouse AP model with decreased Ccl9 expression and less neutrophil accumulation. In conclusion, Cxcl16 expressed in pancreatic acini contributes to the development of acinar cell necrosis through the induction of Ccl9 and subsequent neutrophil infiltration. CXCL16 could be a new therapeutic target in AP.

Aiming to identify factors causing the adverse health effects associated with moisture-damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture-damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n = 25) collected from moisture-damaged and reference schools in Spain, the Netherlands, and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor-α (TNF-)α, interleukin (IL)-6, and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis, and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers such as ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins, and glucans were measured as markers of exposure. Dust from moisture-damaged schools in Spain and the Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram-positive bacteria., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: The purpose of this study was to study the expression of CCL15 in hepatocellular carcinoma (HCC) and explore its clinicopathological significance, and study relationships between expressions of CCL15 and malignant behaviors of HCC., Methods: The SP immunohistochemical method was used to detect expression of CCL15 in routinely paraffin-embedded sections from 80 cases of HCC, 80 of adjacent cancerous specimens and 50 of normal liver tissue. In these patients with HCC, Kaplan-Meier was used to assess survival outcomes., Results: The positive rates and scores of CCL15 were significantly higher in HCC than adjacent cancerous specimens and normal liver tissue (p < 0.05), but not significantly higher between adjacent cancerous specimens and normal liver tissue (p > 0.05). The expression of CCL15 was significantly correlated to tumor size, tumor thrombi in portal vein of HCC, capsule and TNM stage (p < 0.05), but not to sex, age, liver cirrhosis and the level of AFP so on (p > 0.05). Survival time of the patients with positive CCL15 expression was significantly decreased, and multivariate analysis indicated CCL15 expression was one of the independent predictors of survival (p = 0.042)., Conclusion: The expression of CCL15 was significantly correlated with malignant behaviors of HCC, and CCL15 might be important biological markers for reflecting the carcinogenesis, progression, biological behaviors and prognosis of HCC.

Background: Encapsulating peritoneal sclerosis (EPS) is a rare but devastating complication of long-term peritoneal dialysis (PD). There is no well-validated method for predicting which patients will develop the condition, although known risk factors include long duration of PD, high glucose exposure and lack of residual renal function. We have investigated whether dialysate cytokines (MCP-1 (monocyte chemotactic protein-1), CCL18 (pulmonary and activation-regulated cytokine, PARC), IL-6 (interleukin-6), CCL15 (leukotactin) and angiogenin) could be used to predict the onset of EPS more effectively than known clinical risk factors., Methods: Samples of dialysate and clinical data were prospectively collected from 151 patients at the West London Renal center between 2003 and 2010. Dialysate cytokine levels were measured using the enzyme-linked immunoabsorbant assay (ELISA) technique. Encapsulating peritoneal sclerosis subsequently developed in 17 patients during a follow-up period of 27 - 113 months. Cytokines found at higher levels in dialysate of pre-EPS patients were investigated as candidate predictors of EPS using logistic regression analysis., Results: Dialysate IL-6, MCP-1 and CCL15 were significantly higher in patients who subsequently developed EPS; however, a logistic regression model using dialysate cytokines to predict EPS was no better than a model using well-recognized clinical markers (length of time on PD and membrane transport status)., Conclusions: Although MCP-1, IL-6 and CCL15 were found at higher levels in the dialysate of patients who subsequently developed EPS, dialysate levels of these cytokines do not improve prediction of future EPS above a model using known clinical risk factors., (Copyright © 2014 International Society for Peritoneal Dialysis.)

Objective: The differential diagnosis between preterm and false labour remains one of the most challenging issues in perinatal medicine., Aim: To assess the prognostic importance of the selected biochemical markers in predicting preterm labour., Material and Methods: 74 patients hospitalized due to threatening preterm labour. 51 women gave birth prematurely; the remaining 23 were diagnosed with false labour. We used ELISA arrays to study 13 proteins: IGFBP-1, IGFBP-2, BDNF, L-Selectin, E-Selectin, ICAM-1, PECAM, VCAM-1, MIP-1 delta (MIP-1d) MIP-3β (MIP-3b), Eotaxin-1, Eotaxin-2, BLC., Results: An increased risk of preterm labour should be expected when the serum concentration for: IGFBP-1 > 158.83 pg/ml (sens. 0.608, sp. 0.609, p < 0.0001); MIP-1d < 27.66 pg/ml (sens. 0.627, sp. 0.627, p = 0.021); BDNF >36.54 pg/ml (sens. 0.630, sp. 0.647, p = 0.002); BLC >25.46 pg/ml (sens. 0.588, sp. 0.609, p < 0.001); Eotaxin-1 >1.16 pg/ml (sens. 0.633, sp. 0.652)., Conclusion: There have been reported statistically significant differences in serum concentrations of selected proteins in women with preterm labour and false labour.

Airway inflammation is accompanied by infiltration of inflammatory cells and an abnormal response of airway smooth muscle. These cells secrete chemokines and express the cell surface chemokine receptors that play an important role in the migration and degranulation of inflammatory cells. Omalizumab is a monoclonal antibody directed against immunoglobulin E, and its blocking of IgE signaling not only reduces inflammatory cell infiltration mediated by the Th2 immune response but also inhibits other immune responses. The chemokine CCL15 is influenced by omalizumab, and the source of CCL15 has been reported to be airway smooth muscle cells and basophils. CCL15 binds to its receptor CCR1, which has been reported to be expressed by various inflammatory cells and also by airway smooth muscle cells. Therefore, CCL15/CCR1 signaling could be a target for the treatment of asthma. We review the role of CCL15 in the pathogenesis of asthma and also discuss the influence of IgE-mediated immunomodulation via CCL15 and its receptor CCR1.

Omalizumab is an anti-IgE monoclonal antibody that was proven effective for the treatment of severe asthma. IgE plays a central role in allergic asthma, and an anti-allergic effect of omalizumab has been confirmed in terms of its impact on Th2 cytokines. The objective of the present study is to determine the influence of omalizumab on clinical parameters and circulating immuoregulatory cytokines. Patients with severe allergic asthma were enrolled and given four months of omalizumab therapy. Changes of symptoms and other parameters were assessed, including the asthma control test (ACT) score, morning peak expiratory flow (PEF), peripheral eosinophil count, total serum IgE, and pulmonary function tests. The use of corticosteroids and short-acting bronchodilators, as well as the number of unscheduled hospital visits, were monitored. Circulating levels of cytokines were analyzed with a multiplex cytokine immunoassay in patients with or without omalizumab therapy. Asthma symptoms (evaluated by the ACT score and morning PEF) improved with omalizumab treatment, while total IgE was elevated. Use of corticosteroids and short-acting bronchodilators and the number of unscheduled hospital visits for exacerbation of asthma were all reduced by omalizumab treatment. The level of macrophage inflammatory protein 1-δ (MIP1-δ) was significantly reduced after omalizumab therapy and was high in patients without omalizumab. IL-16 also tended to decrease with omalizumab therapy. Both MIP1-δ and IL-16 decreased as asthma improved over the 4-month period of omalizumab therapy. These findings suggest that omalizumab may act via IgE-mediated immunoregulation of MIP1-δ and IL-16.

Background and Objective: Porphyromonas gingivalis infection is thought to be a significant etiological factor in the development of cardiovascular diseases. However, scant definitive evidence has been presented concerning the pathological molecular mechanisms of these disorders. In the present study, we performed a molecular analysis of the developmental mechanisms of aortic intimal hyperplasia induced by P. gingivalis., Material and Methods: The effects of P. gingivalis-induced bacteremia on intimal hyperplasia were evaluated using a mouse model of aortic hyperplasia created by photochemical-induced endothelial cell injury. Alterations of gene expression profiles in injured blood vessels of the mice were extensively analyzed using DNA microarray assays to identify the key molecules involved in P. gingivalis-induced hyperplasia. In addition, human aneurismal specimens from patients with or without P. gingivalis infection were analyzed histochemically., Results: Intravenous administration of P. gingivalis dramatically induced intimal hyperplasia in the mouse model. Concomitantly, S100 calcium-binding protein A9 (S100A9) and embryonic isoform of myosin heavy chain (SMemb), a proliferative phenotypic marker of smooth muscle cells, were significantly overexpressed on the surfaces of smooth muscle cells present in the injured blood vessels. Similarly, increased expressions of S100A9 and SMemb proteins were observed in aneurismal specimens obtained from P. gingivalis-infected patients., Conclusion: We found that bacteremia induced by P. gingivalis leads to intimal hyperplasia associated with overexpressions of S100A9 and SMemb. Our results strongly suggest that oral-hematogenous spreading of P. gingivalis is a causative event in the development of aortic hyperplasia in periodontitis patients.

Ciprofloxacin, the most widely used totally synthetic antibiotic, is subject to active efflux mediated by a MRP-like transporter in wild-type murine J774 macrophages. To identify the transporter among the seven potential Mrps, we used cells made resistant to ciprofloxacin obtained by long-term exposure to increasing drug concentrations (these cells show less ciprofloxacin accumulation and provide a protected niche for ciprofloxacin-sensitive intracellular Listeria monocytogenes). In the present paper, we first show that ciprofloxacin-resistant cells display a faster efflux of ciprofloxacin which is inhibited by gemfibrozil (an unspecific MRP inhibitor). Elacridar, at a concentration known to inhibit P-glycoprotein and breast cancer resistance protein (BCRP), only slightly increased ciprofloxacin accumulation, with no difference between resistant and wild-type cells. Analysis at the mRNA (real-time PCR) and protein (Western blotting) levels revealed an overexpression of Mrp2 and Mrp4. Mrp4 transcripts, however, were overwhelmingly predominant (45% [wild-type cells] to 95% [ciprofloxacin-resistant cells] of all Mrp transcripts tested [Mrp1 to Mrp7]). Silencing of Mrp2 and Mrp4 with specific small interfering RNAs showed that only Mrp4 is involved in ciprofloxacin transport in both ciprofloxacin-resistant and wild-type cells. The study therefore identifies Mrp4 as the most likely transporter of ciprofloxacin in murine macrophages but leaves open a possible common upregulation mechanism for both Mrp4 and Mrp2 upon chronic exposure of eukaryotic cells to this widely used antibiotic.

The objective of this study was to assess protein levels for candidate cytokines, chemokines, growth factors, matrix metalloproteinases and their inhibitors in bronchoalveolar lavage fluid (BALF) in patients with polar forms of pulmonary sarcoidosis, i.e. Löfgren's syndrome (LS) and more advanced chest X-ray (CXR) stage III disease. Twenty-four inflammatory molecules were analysed in unconcentrated BALF samples from 10 sarcoidosis patients with CXR stage III and 10 patients with LS by semiquantitative protein array. Four novel molecules [CC chemokine ligand (CCL)15, CCL16, macrophage migration inhibitory factor (MIF) and macrophage stimulating protein (MSP)], detected for the first time in association with sarcoidosis, were then quantified by enzyme-linked immunosorbent assay in a second cohort of 68 sarcoidosis patients and 17 control subjects. The protein levels of CCL15, CCL16, CCL24, CXCL8, CXCL9, CXCL10, interleukin-16, MIF, MSP and matrix metallopeptidase 1 were increased in CXR stage III patients when compared with patients with LS. CCL15 and MSP up-regulation in CXR stage III patients in comparison with LS patients and controls was confirmed by enzyme-linked immunosorbent assay. Moreover, MSP was associated with treatment requirement (P = 0.001) and CCL15 was elevated in patients with disease progression at 2-year follow-up (P = 0.016). CCL16 levels were increased in sarcoidosis versus controls (P < 0.05), but no difference was observed between patient subgroups. MIF up-regulation was not confirmed in a larger patient group. In conclusion, chemokines CCL15, CCL16 and MSP were found elevated for the first time in BALF from sarcoidosis patients; our results showed that CCL15 and MSP may affect disease course.

Unlabelled: The objectives of this study were to test the feasibility of measuring inflammatory and nociceptive biochemical mediators at the surgical site and to evaluate the relationship between wound and serum levels as well as determine any associations between mediator release, pain, and analgesic consumption after cesarean delivery. Twenty healthy women undergoing elective cesarean delivery with spinal anesthesia were enrolled. Wound exudate and serum mediators, pain scores, and analgesic consumption were measured at 1, 6, 24, and 48 hours after cesarean. In wound exudate, 19 of 20 mediators were reliably detected including interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, tumor necrosis factor-alpha, interferon-gamma, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 (MIP-1beta), nerve growth factor (NGF), prostaglandin E2 (PG-E2), and substance P. Wound PG-E2 and various cytokines peaked early, whereas NGF showed a more delayed release. There were no correlations between the concentration versus time profile of wound and serum cytokines. Analgesic consumption during the first 24 hours after surgery was negatively correlated with IL-1beta, IL-6, and G-CSF in the wound exudate. This study demonstrates the feasibility of collecting and measuring nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between wound and serum levels emphasizes the importance of determining site-specific release if localized pathologies are to be studied., Perspective: This study demonstrates the feasibility of measuring real-time nociceptive and inflammatory mediators in surgical wounds. Our findings confirm the lack of correlation between wound and serum levels of many pro-inflammatory and anti-inflammatory cytokines and nerve growth factor.

Bronchiolitis obliterans syndrome (BOS) is one of the most important factors limiting the long-term survival of lung transplant recipients (LTR), however its pathogenesis still remains unclear. We hypothesized that an increased production of certain specific proinflammatory mediators in the first post-transplant year would predispose to BOS. We retrospectively evaluated temporal kinetics of some CC chemokines that have not yet been evaluated, including CCL3/MIP1-alpha, CCL4/MIP1-beta, CCL17/TARC, CCL19/MIP3-beta, CCL20/MIP3-alpha, CCL22/MDC and CCL26/eotaxin, in broncho-alveolar lavage fluid (BAL-f) in the first post-transplant year in a cohort of 8 LTR before the development of BOS (pre-BOS LTR) and 8 LTR with long-term stable clinical conditions (stable LTR). Chemokine levels were assayed by means of a multiplex sandwich ELISA. Furthermore, for those ligands which resulted significantly predictive of BOS onset, we analyzed the expression of specific receptors (CCR) on BAL cells. The proportion of CCR-expressing BAL cells was assessed by flow cytometry. We demonstrated that MIP3-beta/CCL19, MIP3-alpha/CCL20, MDC/CCL22 levels at 6 months post-transplant significantly predicted BOS onset. In addition, the temporal behavior of these factors resulted significantly different in pre-BOS patients as compared to stable LTR. Finally the expression of CCR was documented on BAL lymphocytes and macrophages, and, in some cases, their expression was found to vary between the two groups. Within the complexity of the chemokine network, these three CCL factors could play an additive role in the pathogenesis of the inflammatory process leading to bronchiolar fibro-obliteration.

Object: Inhibition of remyelination is part of the complex problem of persistent dysfunction after spinal cord injury (SCI), and residual myelin debris may be a factor that inhibits remyelination. Phagocytosis by microglial cells and by macrophages that migrate from blood vessels plays a major role in the clearance of myelin debris. The object of this study was to investigate the mechanisms underlying the failure of significant remyelination after SCI., Methods: The authors investigated macrophage recruitment and related factors in rats by comparing a contusion model (representing contusive SCI with residual myelin debris and failure of remyelination) with a model consisting of chemical demyelination by lysophosphatidylcholine (representing multiple sclerosis with early clearance of myelin debris and remyelination). The origin of infiltrating macrophages was investigated using mice transplanted with bone marrow cells from green fluorescent protein-transfected mice. The changes in levels of residual myelin debris and the infiltration of activated macrophages in demyelinated lesions were investigated by immunostaining at 2, 4, and 7 days postinjury. To investigate various factors that might be involved, the authors also investigated gene expression of macrophage chemotactic factors and adhesion factors., Results: Activated macrophages coexpressing green fluorescent protein constituted the major cell population in the lesions, indicating that the macrophages in both models were mainly derived from the bone marrow, and that very few were derived from the intrinsic microglia. Immunostaining showed that in the contusion model, myelin debris persisted for a long period, and the infiltration of macrophages was significantly delayed. Among the chemotactic factors, the levels of monocyte chemoattractant protein-1 and granulocyte-macrophage colony-stimulating factor were lower in the contusion model at 2 and 4 days postinjury., Conclusions: The results suggest that the delayed infiltration of activated macrophages is related to persistence of myelin debris after contusive SCI, resulting in the inhibition of remyelination.

Background: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to secretion of inflammatory mediators and cytokine production. Basophils are known to play an important role in the pathogenesis of asthma but there has been no comprehensive examination of the effectors these cells produce. Here a study of the transcription and release of a selection of chemokines and cytokines from basophils was undertaken., Methods: A Cartesian antibody array provided an effective method of assaying for multiple cytokines and chemokines simultaneously. Results were verified by RT-PCR and ELISA assays. This allowed the comparison of freshly prepared peripheral blood basophil responses to cross-linking of the high-affinity IgE receptor, with and without preincubation with IL-3., Results: Evidence that human blood basophils produce the chemokines MIP-5, eotaxin and GM-CSF was provided by antibody array and RT-PCR analyses. Preincubation with IL-3 enhanced the expression and release of IL-13, IL-8 and mRNA transcripts encoding MIP-5 and GATA2 in basophils from both asthmatic and control subjects. Leptin mRNA transcription, storage and release in basophils are described for the first time., Conclusions: Surveying cytokine and chemokines stored and released by peripheral blood basophils shows that asthmatic and control subjects share similar profiles even when their degranulation responses are distinct. Evidence is provided for the production of leptin, GM-CSF, eotaxin and MIP-5 by peripheral blood basophils. IL-3 preincubation enhances the production and release of IL-8 upon IgE receptor cross-linking., (2007 S. Karger AG, Basel)

Object: Subarachnoid hemorrhage (SAH) results in the expression of inflammatory and extracellular matrix (ECM)-related genes and various G protein-coupled receptors. In the present study, the authors evaluated the time course and sequence of the transduction pathways, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase-1 and 2 (ERK1/2), and associated transcription factor activation as well as gene regulation and associated protein levels., Methods: Subarachnoid hemorrhage was induced in rats by injecting 250 microl of blood into the suprachiasmatic cistern, and gene regulation in the cerebral arteries was examined at various points in time following SAH by using quantitative polymerase chain reaction (PCR) and immunohistochemistry., Results: Immunohistochemical findings demonstrated that SAH phosphorylates and activates p38 and ERK1/2 as well as the downstream transcription factors Elk-1 and activating transcription factor-2. The pattern of activation consists of a rapid phase within the first few hours and a late phase that occurs from 24 to 48 hours. Activation is followed by an increase in the transcription of the inflammatory and ECM-related genes (IL6, TNFalpha, IL1beta, CXCL1, CXCL2, CCL20, MMP8, MMP9, MMP13, and iNOS), as demonstrated using real-time PCR. For MMP13 and iNOS, the changes in transcription were translated into functional proteins, as revealed on immunohistochemistry., Conclusions: Activation of the p38 and ERK1/2 signaling pathways and their downstream transcription factors can explain the increase in the transcription of the genes studied. This increase and the subsequent augmentation in protein levels suggest that the inflammatory response may in part explain the remodeling that occurs in cerebral arteries following SAH.

We have previously shown that intratracheal instillation of carbon nanoparticles exacerbates lung inflammation related to bacterial endotoxin (lipopolysaccharide, LPS) and subsequent systemic inflammation with coagulatory disturbance in mice [Inoue, K., Takano, H., Yanagisawa, R., Hirano, S., Sakurai, M., Shimada, A., Yoshikawa, T., 2006b. Effects of airway exposure to nanoparticles on lung inflammation induced by bacterial endotoxin in mice. Environ. Health Perspect. 114, 1325-1330]. The present study was performed to determine whether inhalation of diesel engine-derived nanoparticles also exacerbates the model. ICR mice were exposed for 5h to clean air or diesel engine-derived nanoparticles at a concentration of 15, 36, or 169 microg/m(3) after intratracheal challenge with 125 microg/kg of LPS or vehicle, and were sacrificed for evaluation 24h after the intratracheal challenge. Nanoparticles alone did not induce lung inflammation. Nanoparticle inhalation increased LPS-elicited inflammatory cell recruitment into the bronchoalveolar lavage fluid and lung parenchyma as compared with clean air inhalation in a concentration-dependent manner. Lung homogenates derived from the nanoparticle+LPS groups tended to have increased tumor necrosis factor-alpha level and chemotaxis activity for polymorphonuclear leukocytes as compared to those from the LPS group or the corresponding nanoparticle groups. Nanoparticle inhalation did not significantly increase lung expression of proinflammatory cytokines or facilitate systemic inflammation and coagulatory disturbance. Isolated alveolar macrophages (AMs) from nanoparticle-exposed mice showed greater production of interleukin-1beta and keratinocyte chemoattractant stimulated with ex vivo LPS challenge than those from clean air-exposed mice, although the differences did not reach statistical significance. These results suggest that acute exposure to diesel nanoparticles exacerbates lung inflammation induced by LPS.

Objective: The present study describes intercalated disc remodeling under both protein expression and structural features in experimental severe sepsis induced by cecal ligation and puncture in mice., Design: Controlled animal study., Setting: University research laboratory., Subjects: Male C57BL/6 mice., Interventions: Mice were submitted to moderate and severe septic injury by cecal ligation and puncture., Measurement and Main Results: Severe septic injury was accompanied by a large number of bacteria in the peritoneal cavity and blood, high levels of tumor necrosis factor-alpha, and monocyte inflammatory protein-1alpha in the septic focus and serum, marked hypotension, and a high mortality rate. Western blot analysis and immunofluorescence showed a marked decrease of key gap and adherens junction proteins (connexin43 and N-cadherin, respectively) in mice submitted to severe septic injury. These changes may result in the loss of intercalated disc structural integrity, characterized in the electron microscopic study by partial separation or dehiscence of gap junctions and adherens junctions., Conclusions: Our data provide important insight regarding the alterations in intercalated disc components resulting from severe septic injury. The intercalated disc remodeling under both protein expression and structural features in experimental severe sepsis induced by cecal ligation and puncture may be partly responsible for myocardial depression in sepsis/septic shock. Although further electrophysiological studies in animals and humans are needed to determine the effect of these alterations on myocardial conduction velocity, the abnormal variables may emerge as therapeutic targets, and their modulation might provide beneficial effects on future cardiovascular outcomes and mortality in sepsis.

Breastmilk chemokines have been associated with increased HIV-1 RNA levels in breastmilk and altered risk of mother-to-child HIV-1 transmission. To characterize CC and CXC chemokines in breastmilk postpartum, we collected breastmilk specimens at regular intervals for 6 months after delivery from women with and without HIV-1 infection and used commercial ELISA kits to measure breastmilk concentrations of MIP-1alpha, MIP-1beta, RANTES, and SDF-1alpha. Among 54 HIV-1-infected and 26 uninfected women, mean chemokine levels were compared cross-sectionally and longitudinally at days 5 and 10, and months 1 and 3 postpartum. For both HIV-1-infected and uninfected women, breastmilk chemokine levels were highest at day 5 for MIP-1alpha, MIP-1beta, and SDF-1alpha, and subsequently decreased. RANTES levels remained constant over the follow-up period among HIV-1-uninfected women, and increased moderately among HIV-1-infected women. For MIP-1beta and RANTES, breastmilk levels were significantly higher among HIV-1-infected women compared to uninfected women early postpartum. In addition, HIV-1-infected women transmitting HIV-1 to their infant had consistently higher breastmilk RANTES levels than those who did not transmit, with the greatest difference observed at 1 month (2.68 vs. 2.21 log10 pg/mL, respectively; p = 0.007). In summary, all four chemokines were most elevated within the first month postpartum, a period of high transmission risk via breastmilk. MIP-1beta and RANTES levels in breastmilk were higher among HIV-1-infected women than among uninfected women, and breastmilk RANTES was positively associated with vertical transmission in this study, consistent with results from our earlier cohort.

Cystic fibrosis (CF) is the commonest multisystem genetic disease of white races, caused by mutations in the cystic fibrosis transmembrane regulator (CFTR), encoded on the long arm of chromosome 7. Mutations in the CFTR gene result in defective sodium, chloride, and water transport in the epithelial cells of the respiratory, hepatobiliary, gastrointestinal, and reproductive tracts, the pancreas, and the eye. The pathogenesis of ocular changes in CF is still unknown, but CF belongs to the large pathologic group of ocular surface epithelial diseases, termed keratoconjunctivitis sicca (KCS), that develop in dry eye syndrome. The aim of this study was to evaluate the levels of macrophage inflammatory protein-1alpha (MIP-1alpha) in the tear fluid of CF patients. We also investigated the correlation between the tear levels of this chemokine and clinical severity of CF and ocular surface disease. We studied 25 patients with CF with a mean age of 14 years. Chemokine levels were determined by ELISA. Complete ophthalmic examination, including dry eye tests, were used to study the ocular surface. The tear levels of MIP-1alpha in the CF patients were significantly higher when compared with healthy controls. We found a negative correlation between the tear levels of MIP-1alpha and clinical severity in CF patients and a positive correlation between the tear levels of MIP-1alpha and the presence of dry eye findings in CF patients. This current study indicates that chemokines play an important role in the ongoing inflammatory response. Our findings may help to explain one of the key factors contributing to the pathogenesis of ocular surface changes in CF patients.

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. It is unclear if dendritic cells (DC) participate in this inflammatory process., Objectives: To evaluate the presence of DC in small airways of patients with COPD., Methods: We evaluated DC infiltration in small airways by immunohistochemistry in patients with COPD (stage I-IV), never-smokers, and smokers without COPD. Chemokine ligand 20 (CCL20, the most potent chemokine in attracting DC) was determined in total lung by RT-PCR and in induced sputum by enzyme-linked immunsorbent assay. Chemokine receptor 6 (CCR6, the receptor for CCL20) expression on human pulmonary DC was evaluated by RT-PCR and flow cytometry., Measurements and Main Results: There is a significant increase in DC number in the epithelium (p = 0.007) and adventitia (p = 0.009) of small airways of patients with COPD compared with never-smokers and smokers without COPD. DC number in epithelium and adventitia increases along with disease severity. CCL20 mRNA expression in total lung and CCL20 protein levels in induced sputum are significantly higher in patients with COPD compared with never-smokers (p = 0.034 for CCL20 mRNA and p = 0.0008 for CCL20 protein) and smokers without COPD (p = 0.016 for CCL20 mRNA and p = 0.001 for CCL20 protein). DC isolated from human lung express CCR6 both at mRNA and at protein level., Conclusions: This is the first description of airway infiltration by DC in COPD. Moreover, interaction between CCL20 and CCR6 provides a possible mechanism for accumulation of DC in the lungs in COPD.

Suppression of immune activation and increased inflammation are prevalent during viral infection. To investigate the role of inflammation in HIV transmission, we studied the infectious and inflammatory milieu in cervical mucosa from HIV-1- and human papillomavirus (HPV)-coinfected and HPV-monoinfected women. The numbers of cytokine-, chemokine-, and p24-expressing cells were determined using in situ imaging analysis and intracellular staining of p24 antigen. Significantly higher expression of the proinflammatory cytokines, interleukin (IL)-1alpha/beta, was seen in cervical tissue from HIV/HPV-coinfected as compared with HPV-monoinfected tissues, whereas IL-2- and interferon (IFN)-gamma-expressing cells were higher in HPV-monoinfected tissues. IL-10 was low in both groups, whereas IL-4 was significantly higher in HPV-monoinfected and HIV/HPV-coinfected tissues than in HIV/HPV-negative controls. RANTES and macrophage inflammatory protein (MIP)-1beta but not MIP-1alpha were significantly higher in the genital tract of HIV/HPV-coinfected as compared with HPV-monoinfected individuals and controls. HIV/HPV-coinfected tissues had a higher level of human leukocyte antigen D-related (HLA-DR)-expressing dendritic cells (DCs). There was a positive correlation between the number of CD4(+) and CD8(+) T cells as well as CD1a, IL-1alpha, and RANTES expression and p24 antigen-expressing cells in the HIV/HPV-coinfected tissues. These findings suggest the persistence of immune activation and inflammation in the genital tract of women with HPV monoinfection and in HIV-infected women coinfected with HPV.

Acute expression of macrophage inflammatory protein-1 beta (also known as CCL4) promotes beneficial leukocyte recruitment to infected tissues, but chronic expression of this chemokine contributes to inflammatory disease. CCL4 expression is controlled largely at the transcriptional level and an ATF/CRE sequence located in the promoter (-104 to -97bp, relative to the transcriptional start site) has been identified as a critical cis-acting element. The trans-acting binding proteins that influence CCL4 transcription via this site are largely unknown. We investigated whether activating transcription factor 3 (ATF3), a member of the ATF/CREB family of transcription factors, binds to the CCL4 ATF/CRE site in macrophages. Using the electrophoretic mobility shift assay and the chromatin immunoprecipitation assay, we found that ATF3 binds to the ATF/CRE site within the CCL4 promoter in untreated and lipopolysaccharide (LPS)-stimulated macrophages. Quantitative RT-PCR analysis showed that CCL4 mRNA levels in elicited peritoneal macrophages from ATF3(-/-) mice are significantly higher than in congenic ATF3(+/+) macrophages under both unstimulated and LPS-stimulated conditions, suggesting that ATF3 represses transcription of the CCL4 gene. Consistent with the higher gene expression, ATF3-deficient macrophages secreted more CCL4 protein than ATF3(+/+) macrophages. Similar results were obtained in bone-marrow-derived macrophages treated with Toll-like receptor 2, 3, 4 and 5 agonists. Thus, we conclude that ATF3 constitutively binds to the ATF/CRE site in the CCL4 promoter where it represses basal and pathogen-associated molecular pattern (PAMP)-stimulated transcription. Consequently, ATF3 appears to be part of a control mechanism that limits the amount of CCL4 released by macrophages, preventing excessive inflammation.

Background: Patients with atopic dermatitis (AD) are prone to disseminated viral skin infections and therefore are not vaccinated against smallpox because of potential complications. Macrophage inflammatory protein 3alpha (MIP-3alpha) is a C-C chemokine expressed by keratinocytes that exhibits antimicrobial activity against bacteria and fungi; however, its role in antiviral innate immunity is unknown., Objective: Evaluate the level of MIP-3alpha in AD skin and its role in the innate immune response to vaccinia virus (VV)., Methods: Macrophage inflammatory protein 3alpha levels were evaluated using real-time RT-PCR, immunodot-blot, and immunohistochemistry. The antiviral activity of MIP-3alpha was determined using a standard viral plaque assay., Results: Macrophage inflammatory protein 3alpha gene expression was significantly (P < .01) decreased in AD skin (0.21 +/- 0.05 ng MIP-3alpha/ng glyceraldehyde-3-phosphate dehydrogenase) compared with psoriasis skin (0.67 +/- 0.13). This was confirmed at the protein level using immunohistochemistry. We further demonstrate that T(H)2 cytokines downregulate MIP-3alpha expression. The importance of MIP-3alpha in the innate immune response against VV was established by first demonstrating that MIP-3alpha exhibits activity against VV. Second, VV replication was significantly increased (P < .01) in keratinocytes treated with an antibody to neutralize MIP-3alpha., Conclusion: The current study demonstrates that MIP-3alpha exhibits antiviral activity against VV and demonstrates the importance of MIP-3alpha in the innate immune response against VV. In addition, AD skin is deficient in MIP-3alpha, in part because of the overexpression of T(H)2 cytokines in AD skin., Clinical Implications: MIP-3alpha deficiency in AD skin contributes to patients' increased propensity toward eczema vaccinatum. Increasing MIP-3alpha or neutralizing T(H)2 cytokines could prevent adverse reactions in patients with AD after smallpox vaccination.

Odontoblasts (OBs) are cells lining the inner surface of the tooth. Their potential role in host defenses within the tooth is suggested by their production of antimicrobial beta-defensins, but their role needs confirmation. The present study sought to define the roles of human OBs in microbial recognition and innate host responses. Toll-like receptor 2 (TLR2) and TLR4, as well as CCR6, were immunolocalized in human OBs and their dentinal processes in situ. To examine OB function we used organotypic tooth crown cultures to maintain human OBs within their dentin scaffold. Cells in the OB layer of cultured and non-cultured crown preparations expressed mRNA for several markers of innate immunity including chemokine CCL20, chemokine receptor CCR6, TLR2, TLR4 and the OB marker dentin sialophosphoprotein (DSPP). Expression of human beta-defensin 1 (hBD1), hBD2, hBD3, interleukin-8 (IL-8), and CCL20 increased with time in culture. Tooth crown odontoblast (TcOB) cultures were stimulated with agonist that was specific for TLR2 (Pam3CSK4) or TLR4 [Escherichia coli lipopolysaccharide (LPS)]. Nuclear factor-kappaB assays confirmed the TLR2 activity of Pam3CSK4 and the TLR4 activity of LPS. LPS up-regulated IL-1beta, tumor necrosis factor-alpha (TNF-alpha), CCL20, hBD2, IL-8, TLR2 and TLR4; however, Pam3CSK4 down-regulated these mRNAs. IL-1beta, TNF-alpha, CCL20 were also up-regulated from six-fold to 30-fold in TcOB preparations from decayed teeth. Our results show for the first time that OBs express microbial pattern recognition receptors in situ, thus allowing differential responses to gram-positive and gram-negative bacteria, and suggest that pro-inflammatory cytokines and innate immune responses in decayed teeth may result from TLR4 signaling.

Objectives: Temporomandibular joint disorders (TMD) comprise a group of chronic painful conditions of mastication in the temporomandibular joint (TMJ). Although the association between TMD and internal derangement of the TMJ is well documented, the functional relevance is still unclear. Increased concentrations of inflammatory mediators have been identified in the synovial fluid of affected patients with TMD, suggesting an underlying degenerative or inflammatory process. The aim of this study was to generate a comprehensive cytokine expression profile in TMD., Methods: 15 samples from patients with internal derangement of TMJ were analysed using a novel cytokine array that enables the analysis of 79 different cytokines simultaneously., Results: Cytokine levels were correlated with the presence of joint effusion (JE) determined by MRI. In the majority of synovial fluid samples, angiogenin (Ang), fibroblast growth factor (FGF)-9, insulin-like growth factor-binding protein (IGFBP)-3, interleukin (IL)-1alpha, IL-1beta, IL-8, inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1beta, osteoprotegerin (OPG), transforming growth factor (TGF)-beta2, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, tumour necrosis factor (TNF)-beta and vascular endothelial growth factor (VEGF) were detectable. Furthermore, the expression levels of Ang, brain-derived neurotrophic factor (BDNF), FGF-4, FGF-9, IGFBP-2, IL-8, MIP-1beta, OPG, pulmonary and activation-regulated protein (PARC), TGF-beta2, TIMP-2 and VEGF were significantly associated with the presence of JE; among these, nine cytokines (Ang, BDNF, FGF-4, FGF-9, IGFBP-2, MIP-1beta, PARC, TGF-beta2 and TIMP-2) were hitherto not described in TMD., Conclusions: This study confirmed previous reports of elevated cytokine levels in TMD. Additionally, we identified previously undescribed cytokines that were upregulated and correlated significantly with the presence of JE. We were able to identify novel cytokines that have hitherto not been described in TMD. Strategies targeting the identified cytokines may represent a novel therapy option in TMD.

(-)-Epigallocatechin-3-gallate (EGCG) is the major active component of green tea. Increasing evidence has suggested that EGCG exhibits anti-inflammatory, anti-oxidant and immunosuppressive effects. In this study, we investigated the effect of EGCG on concanavalin A (ConA)-induced hepatitis (CIH) in mice, a model of immune-mediated liver injury in humans. We pretreated mice with EGCG before ConA injection, and then measured alanine aminotransferase (ALT) levels in plasma, inflammatory infiltration and hepatocyte apoptosis in liver. Potential therapeutic mechanisms were elucidated further by measuring several inflammatory mediators. Mice pretreated with EGCG exhibited much less increased ALT levels in plasma, reduced inflammatory infiltration and hepatocyte apoptosis in liver compared with control mice pretreated with vehicle solutions. We further investigated the mechanisms of the protective effects of EGCG. In EGCG-pretreated mice, we found abrogated tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma at both protein levels in plasma and mRNA levels in liver. At the same time, the concentration of nitrite in plasma and inducible nitric oxide synthase production in liver were both down-regulated in these mice. Moreover, IFN-inducible protein-10 and macrophage inflammatory protein-1alpha expressions in liver were decreased significantly. Therefore, EGCG is capable of regulating immune-mediated liver injury in vivo. The protective effect depended on its suppressive effect on the production of important inflammatory mediators.

Mice deficient in RNA-dependent protein kinase (PKR-/-) or deficient in PKR and a functional 2',5'-oligoadenylate synthetase (OAS) pathway (PKR/RL-/-) are more susceptible to genital herpes simplex virus type 2 (HSV-2) infection than wild-type mice or mice that are deficient only in a functional OAS pathway (RL-/-) as measured by survival over 30 days. The increase in susceptibility correlated with an increase in virus titre recovered from vaginal tissue or brainstem of infected mice during acute infection. There was also an increase in CD45+ cells and CD8+ T cells residing in the central nervous system of HSV-2-infected PKR/RL-/- mice in comparison with RL-/- or wild-type control animals. In contrast, there was a reduction in the HSV-specific CD8+ T cells within the draining lymph node of the PKR/RL-/- mice. Collectively, activation of PKR, but not of OAS, contributes significantly to the local control and spread of HSV-2 following genital infection.

We explore relationships linking clinical symptoms, respiratory dysfunction, and local production of proinflammatory chemokines in the pneumonia virus of mice (PVM) model of viral bronchiolitis. With a reduced inoculum of this natural rodent pathogen, we observe virus clearance by day 9, while clinical symptoms and respiratory dysfunction persist through days 14 and 17 postinoculation, respectively. Via microarray and ELISA, we identify expression profiles of proinflammatory mediators MIP-1alpha, MCP-1, and MIP-2 that correlate with persistent respiratory dysfunction. MIP-1alpha is localized in bronchial epithelium, which is also the major site of PVM replication. Interferon-gamma was detected in lung tissue, but at levels that do not correlate with respiratory dysfunction. Taken together, we present a modification of our pneumovirus infection model that results in improved survival and data that stand in support of a connection between local production of specific mediators and persistent respiratory dysfunction in the setting of acute viral bronchiolitis.

Autoimmune thyroid diseases are characterized by lymphocytic infiltration of the thyroid gland. Chemokines are crucial in the recruitment of lymphocytes and might play an important role in the pathogenesis of autoimmune thyroid disease. The aim of this study was to test the feasibility of analysing by one-tube reverse-transcriptase polymerase chain reaction (RT-PCR) technique CC chemokine profiles in samples obtained by fine needle aspiration biopsy (FNAB). In 27 out of 35 (77%) samples, the material was sufficient for analysis and in 16 (59%) chemokines were detected, thus demonstrating the potential of this technique. Moreover, even in this small group, a statistically significant increase of CCL3 and CCL4 was found in samples from patients with autoimmune thyroid disease as compared to those with multinodular goiter. Chemokine profile measured by improved multiamplification techniques in FNAB thyroid samples may become a useful complementary tool for the management of thyroid autoimmune disease as it constitutes a source of data for research of their pathogenesis.

Previous studies demonstrated that ozone-induced lung damage and inflammation are much greater in hyperthyroid rats, compared to normal rats, at 18 h postexposure. The purpose of the present investigation was to study early events and mechanisms underlying the increased sensitivity to ozone in a hyperthyroid state. Specifically, the degree of lung epithelial cell barrier disruption, the antioxidant status of the extracellular lining fluid, and the release of inflammatory mediators were examined. To induce a hyperthyroid state, mature male Sprague-Dawley rats were implanted with time-release pellets containing thyroxine; control rats received placebo pellets. After 7 d, the animals were exposed to air or ozone (2 ppm, 3 h). Immediately following the end of the exposure, bronchoalveolar lavage (BAL) fluid and cells were harvested. BAL fluid albumin levels and total antioxidant status were examined. In addition, levels of prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)-2, MCP-1, and tumor necrosis factor (TNF)-alpha were determined in BAL fluid and in media samples following ex vivo culture of BAL cells harvested after in vivo inhalation exposures. The results of this study are consistent with the following hypotheses: (1) A marked increase in the permeability of the alveolar-capillary barrier is an early event following ozone exposure in a hyperthyroid state; however this does not appear to be due to overall changes in BAL fluid antioxidant potential. (2) Early increases in MIP-2, but not PGE2, are involved in the enhanced lung response to ozone in a hyperthyroid state. (3) Inflammatory mediator production (i.e., PGE2, MIP-2, MCP-1, and TNF-alpha) by alveolar macrophages plays a minimal role in the initial responses to ozone in a hyperthyroid state.

Seminal fluid is known to be responsible for orchestrating mating-induced immunomodulation. Central to this process are numerous cytokines that modulate uterine leukocyte recruitment and trafficking. Despite this, a comprehensive analysis of the cytokine profile of murine seminal fluid is lacking. This study addressed this issue by using multiplex immunoassays to characterise the profile of interleukin (IL)-1alpha , IL-1beta , IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-gamma, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha , MIP-1beta , regulated upon activation normal T-cell expressed and secreted (RANTES), and tumour necrosis factor (TNF)-alpha in fluid drawn from the seminal vesicles of single mice (n = 18). Their levels and ratios were compared with those found in serum. IL-1alpha , IL-1beta , IL-2, IL-5, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-17, GM-CSF, IFN-gamma, MCP-1 and TNF-alpha levels were significantly higher in serum; IL-4, G-CSF, eotaxin, KC and RANTES exhibited the opposite trend. Based on these findings, we propose a model of mating-induced immunomodulation that implicates seminal eotaxin, RANTES and MIP-1alpha in the relocation and concentration of extravasated migrating endometrial eosinophils to the luminal epithelium. Furthermore, KC may participate in uterine neutrophil chemotaxis and activation. Eotaxin and MIP-alpha , together with IL-1beta and IL-9, may also enhance further cytokine synthesis for endometrial antigen-presenting cell recruitment for processing paternal ejaculate antigens. IL-4 and G-CSF could also minimise deleterious cell-mediated immunity and modulate IFN-gamma production, thereby supporting the establishment of pregnancy.

CCL20 is expected to play a crucial role in the initiation of immune responses and tumour growth. However, expression of CCL20 in Epstein-Barr virus (EBV)-associated diseases has not been studied. We examined the contribution of EBV infection and EBV-encoded latent membrane protein (LMP)-1 to CCL20 expression. EBV infection and LMP-1 induced CCL20 mRNA expression in the EBV-negative Burkitt lymphoma (BL) cell lines and the embryonic kidney cell line. Histone deacetylase inhibitor-stimulated endogenous LMP-1 also induced CCL20 expression in an EBV-positive BL cell line. Analysis of the CCL20 promoter showed that it was activated by LMP-1 C-terminal activation region (CTAR)-1 and CTAR-2. Co-expression of IkappaB alpha, IkappaB beta, IkappaB kinase (IKK)alpha, IKKbeta, IKKgamma, nuclear factor (NF)-kappaB-inducing kinase and tumour necrosis factor receptor-associated factor 2 dominant-negative constructs with LMP-1 inhibited the activation of the CCL20 promoter by LMP-1, suggesting that LMP-1 induces CCL20 via NF-kappaB signalling. The requirement for the NF-kappaB-binding site in the CCL20 promoter in LMP-1 responsiveness was established. Our results indicate that activation of the NF-kappaB pathway by LMP-1 is required for the activation of CCL20 expression.

Background and Aims: Activation of T cells by dendritic cells (DC) is thought to play a pivotal role in induction and maintenance of Crohn's disease. Detailed analyses however concerning the phenotype and maturation of DC as well as the mechanisms underlying their recruitment are still lacking for Crohn's disease., Methods: Different myeloid and plasmacytoid DC subsets were characterised by immunohistochemistry. Expression of the so-called "lymphoid" chemokines CCL19, CCL20, and CCL21 was determined by real time reverse transcription-polymerase chain reaction in Crohn's disease and normal controls. Furthermore, expression of CCL19, CCL20, and CCL21 as well as their receptors CCR6 (for CCL20) and CCR7 (for CCL19 and CCL21) was characterised by immunohistochemistry and, in addition, their cellular localisation was determined by double immunofluorescence investigations., Results: Colonic tissue affected by Crohn's disease was characterised by an increased number of mature myeloid DC forming clusters with proliferating T cells. In keeping with their advanced maturation, DC possess the chemokine receptor CCR7. Increased expression of the CCR7 ligands CCL19 by DC themselves as well as CCL21 by reticular cells and lymphatic vessels was observed in Crohn's disease, thereby causing the matured DC to be trapped at the site of inflammation., Conclusion: Our results demonstrate that autocrine and paracrine actions of lymphoid chemokines in Crohn's disease may lead to increased numbers of mature DC away from their usual migration to lymphoid organs and result in the development of a tertiary lymphatic tissue within the bowel wall maintaining the autoimmune inflammation in Crohn's disease.

Background: Steroid treatment of allergic eosinophilic airway diseases is considered to attenuate cell recruitment by inhibiting several chemokines and to cause eosinophil clearance through inducement of apoptosis of these cells. However, roles of these mechanisms in the actions of steroids in vivo have not been fully established. Also, as regards clearance of tissue eosinophils other mechanisms than apoptosis may operate in vivo., Objective: This study explores anti-inflammatory effects of steroids instituted during either development or resolution of airway allergic inflammation., Methods: Immunized mice were subjected to week-long daily allergen challenges (ovalbumin). Steroid treatment was instituted either amidst the challenges or exclusively post-allergen challenge. CC chemokines, goblet cell hyperplasia, occurrence of eosinophil apoptosis, and airway tissue as well as lumen eosinophilia were examined at different time-points., Results: Daily steroids instituted amid the allergen challenges non-selectively attenuated a range of chemokines, permitted egression of tissue eosinophils into airway lumen to increase, and reduced development of lung tissue eosinophilia. Steroid treatment instituted post-challenge selectively inhibited the CC-chemokine regulation upon activation, normal T cell expressed and secrted (RANTES), permitted continued egression of eosinophils into airway lumen, and resolved the tissue eosinophilia. Eosinophil apoptosis rarely occurred at development and resolution of the allergic eosinophilic inflammation whether the animals were steroid treated or not. However, anti-Fas monoclonal antibodies given to mice with established eosinophilia post-challenge produced apoptosis of the tissue eosinophils indicating that apoptotic eosinophils, if they occur, are well detectible in vivo., Conclusion: Airway tissue eosinophils are likely eliminated through egression into airway lumen with little involvement of apoptosis and phagocytosis. Our data further suggest that therapeutic steroids may resolve airway inflammation by permitting clearance of tissue eosinophils through egression and inhibiting RANTES-dependent cell recruitment to lung tissues.

A major component in green tea, epigallocatechin-3-gallate (EGCG), is reported to interfere with different steps of a number of inflammatory pathways. After oral administration, EGCG is retained in the gastrointestinal tract, where it is thought to exert preventive functions against inflammatory bowel disease and colon cancer. In this study, the human colon adenocarcinoma cell lines HT29 and T84 were used to investigate the effect of EGCG on intestinal inflammation. HT29 and T84 cells were stimulated with tumor necrosis factor (TNF)-alpha to induce the inflammatory condition and to trigger the inflammatory cascade in vitro and treated with EGCG to study its effect on inflammatory processes. The secretion of the chemokines interleukin (IL)-8, macrophage inflammatory protein (MIP)-3alpha, and prostaglandin E2 (PGE2) was determined by enzyme-linked immunosorbent assay. The gene expression level was measured by quantitative real-time polymerase chain reaction. Treatment of TNF-alpha-stimulated HT29 cells with EGCG dose-dependently inhibited the synthesis of IL-8, MIP-3alpha, and PGE2. Treatment with EGCG also inhibited the production of IL-8 and MIP-3alpha in TNF-alpha-stimulated T84 cells. Gene expression analysis in both HT29 and T84 cells revealed that EGCG down-regulates genes involved in inflammatory pathways. This study shows that EGCG acts broadly on the production of chemokines and PGE2 in the chemokine and eicosanoid pathways of colon epithelial cells. Therefore, EGCG might prove useful for the prevention and/or attenuation of colonic disorders.

We investigated the relationships between plasma monocyte chemoattractant protein-1, serum C-reactive protein, and the degree of hepatic inflammation in patients with chronic liver disease. Monocyte chemoattractant protein-1 concentration was correlated with the histological hepatic inflammation (estimated by the Knodell index) and with standard liver function tests (P<0.01). C-reactive protein was not correlated with any of the variables studied. These results underline the role of monocyte chemoattractant protein-1 in the pathogenesis of liver impairment and suggest that this chemokine may be a reliable marker of inflammation in hepatic derangements.

Objectives: The mechanisms of Fas-Fas ligand (Fas-FasL)-mediated apoptosis in the pathogenesis of fulminant hepatic failure (FHF) have not been well defined. This clinical study was carried out to assess which cells expressed Fas-FasL and to determine their involvement., Methods: The subjects were 24 patients with FHF who underwent liver transplantation at our institution. For comparison, nine chronic hepatitis (CH) patients and six living liver donors (LD) were also enrolled. Liver tissues were obtained for histological (hematoxylin-eosin, terminal deoxynucleotidyl transferase [TdT]-mediated dUTP-biotin nick-end labeling [TUNEL], immunohistochemistry, and double immunofluorescence staining) and reverse transcription PCR (RT-PCR; cytokines and chemokines) analysis., Results: The numbers of TUNEL-, FasL-, and CD68-positive cells in the livers of patients with FHF were significantly larger than in those with CH or with normal livers. Double immunofluorescence staining showed that FasL was expressed predominantly on liver macrophages and rarely on CD8-positive lymphocytes. RT-PCR study showed increased expression of FasL; interferon-gamma; interleukin-18; macrophage inhibitory protein-1beta; and regulated upon activation, normal T cell expressed and secreted in the livers of patients with FHF compared with those of LD., Conclusions: Macrophages and their expression of FasL may play roles in the pathogenesis of FHF.

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.

Introduction: CC and CXC chemokines may play a role in mother-to-child HIV-1 transmission by blocking HIV-1 binding to chemokine receptors and impeding viral entry into cells., Methods: To define correlates of breastmilk chemokines and associations with infant HIV-1 acquisition, chemokines in breastmilk and infant HIV-1 infection risk were assessed in an observational, longitudinal cohort study. We measured MIP-1alpha, MIP-1beta, RANTES, and SDF-1 in month 1 breastmilk specimens from HIV-1-infected women in Nairobi and HIV-1 viral load was calculated in maternal plasma and breastmilk at delivery and 1 month postpartum. Infant infection status was determined at birth and months 1, 3, 6, 9, and 12., Results: Among 281 breastfeeding women, 60 (21%) of their infants acquired HIV-1 during follow-up, 39 (65%) of whom became infected intrapartum or after birth. MIP-1alpha, MIP-1beta, RANTES, and SDF-1 were all positively correlated with breastmilk HIV-1 RNA (P<0.0005). Women with clinical mastitis had 50% higher MIP-1alpha and MIP-1beta levels (P<0.001 and P=0.006, respectively) and women with subclinical mastitis (breastmilk Na(+)/K(+)>1) had approximately 70% higher MIP-1alpha, MIP-1beta and RANTES (P<0.002 for all) compared to women without mastitis. Independent of breastmilk HIV-1, increased MIP-1beta and SDF-1 were associated with reduced risk of infant HIV-1 (RR=0.4; 95% CI 0.2-0.9; P=0.03 and RR=0.5; 95% CI=0.3-0.9; P=0.02, respectively) and increased RANTES was associated with higher transmission risk (RR=2.3; 95% CI 1.1- 5.3; P=0.04)., Conclusions: These observations suggest a complex interplay between virus levels, breastmilk chemokines, and mother-to-child HIV-1 transmission and may provide insight into developing novel strategies to reduce infection across mucosal surfaces.

Background: Periapical lesions are thought to be the result of a local inflammatory response mediated by inflammatory cell infiltration and production of inflammatory mediators. Although chemokines are strongly implicated in the migration and activation of leukocytes in different inflammatory diseases and experimental models, little is known regarding the expression of chemokines and their receptors in human apical periodontitis., Objective and Methods: The objective of this study was to determine the expression of chemokines and their receptors by real-time polymerase chain reaction in samples obtained from healthy gingiva, periapical granulomas, and inflammatory periradicular cysts. The inflammatory infiltrate was characterized by immunohistochemistry., Results: Comparing cysts and granulomas, an increase in CD4+ and CD8+ cells was observed in granulomas, despite the similar numbers of CD45RO-positive cells detected in both lesions. The analysis of mRNA expression revealed increased levels of CCR1, CCR2, CCR3, CCR5, CXCR1, and CXCR3 in both types of lesion compared with controls. Cysts exhibited a higher expression of CCR3, CCR5, CXCR1, and CXCR3 compared to granulomas. A significantly higher expression of RANTES, IP-10, and MCP-1 was detected in cysts compared with controls or granulomas. The expression of interleukin-8, MIP-1alpha, and MIP-1beta was not different in the three experimental groups., Conclusions: The increase in Th1 type (CCR1, CCR5, and CXCR3) and Th2 type (CCR2 and CCR3) receptors in both periapical lesions suggests the concomitant occurrence of Th1 and Th2 responses. Furthermore, the prevalent expression of the receptors CCR3, CCR5, CXCR1, and CXCR3 and of the chemokines RANTES, IP-10, and MCP-1 in cysts may point to a role in the progression of granulomas to cysts.

Background: Clinical manifestations of rhinosinusitis include acute rhinosinusitis, chronic rhinosinusitis (CRS) with nasal polyps and CRS without polyps., Objective: Possible mechanisms defining these three forms of rhinosinusitis should be investigated assessing biomarker profiles in nasal secretions., Methods: Fifteen cytokines, three cellular activation markers and total IgE were determined in nasal secretions of seven patients with acute rhinosinusitis, 12 patients with CRS without polyps, 13 patients with CRS with polyps and six healthy controls. Principal component analysis was used to extract relevant factors., Results: Irrespective of the clinical manifestation, all biomarkers assessed were increased in patients with rhinosinusitis when compared with controls (P<0.001). Principal component analysis allowed the extraction of three factors explaining 83% of data variance. The general inflammatory activation was mainly reflected by the first factor. The second factor differentiated acute from CRS. This factor correlated with IL-12, which is involved in pathogen-related immune activation by antigen-presenting cells. It was also positively correlated with IL-4, IL-10 and IL-13, which play an important role in the resolution of infections. The third factor differentiated CRS with polyps from CRS without polyps (P=0.001). It represented IL-5 and nasal IgE (nIgE), whereas eosinophil cationic protein and tryptase were not specific for CRS with polyps., Conclusion: In mucosal infection, numerous inflammatory mediators are activated. Simple correlations of few biomarkers with a specific disease process bear the risk of overestimating a possibly unspecific effect. To assess biomarker profiles, more complex analytic tools may be more appropriate to delineate mechanisms underlying mucosal disease. Using principal component analysis, it was found that high nIgE and IL-5 levels are specific for CRS with nasal polyps.

Background: Periodontal diseases may differ, which could be attributed to the factors that might modify the host response to microbial pathogens. The aim of this study was to examine gingival crevicular fluid (GCF) levels of EMAP-II, MIP-1alpha and MIP-1beta in patients with different periodontal diseases (EMAP-II, endothelial-monocyte activating polypeptide; MIP-1alpha, macrophage inflammatory protein-1alpha; MIP-1beta, macrophage inflammatory protein-1beta)., Methods: Eighty-two subjects were included in this study. GCF samples were collected from 26 patients with generalized aggressive periodontitis (G-AgP), 26 patients with chronic periodontitis (CP), 15 with gingivitis and 15 periodontally healthy subjects. Clinical periodontal parameters were recorded. GCF EMAP-II, MIP-1alpha and MIP-1beta levels were quantified by enzyme immunoassay., Results: GCF EMAP-II levels of G-AgP group were higher than those of gingivitis and healthy groups (p<0.008). G-AgP group showed a trend for higher GCF EMAP-II levels compared with CP group (p>0.008). G-AgP, CP, gingivitis and healthy groups had comparable GCF MIP-1alpha and MIP-1beta levels., Conclusions: Our results suggest that elevated GCF EMAP-II could contribute to the pathogenesis of G-AgP. Alternatively, EMAP-II reflects the extent of the inflammatory activity in the periodontal tissues. At this point, MIP-1alpha and MIP-1beta levels in GCF do not seem to play a discriminatory role in periodontitis. Our data document for the first time the essential role of EMAP-II in the pathogenesis of different periodontal diseases.

In rheumatoid arthritis (RA), a widespread autoimmune/inflammatory joint disease, early activation of effector CD4+ T lymphocytes, and cytokine production is followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, and disease. GITR (glucocorticoid-induced TNFR family-related gene), a costimulatory molecule for T lymphocytes, increases CD4+CD25- effector T cell activation while inhibiting suppressor activity of CD4+CD25+ T regulatory (Treg) cells. We analyzed the role of GITR in type II collagen (CII) -induced arthritis (CIA) using GITR-/- and GITR+/+ mice. Results indicate significantly less CIA induction in GITR-/- mice than in GITR+/+ mice, with marked differences in erythema, edema, neutrophil infiltration, joint injury, and bone erosion. Production of IFNgamma, IL-6, TNFalpha, MIP-1alpha, and MIP-2, inducible NOS (iNOS), COX-2, and nitrotyrosine poly-ADP-ribose (PAR) were also less in CII-treated GITR-/- mice. Although CD4+CD25+ Treg cells from GITR+/+ and GITR-/- CII-challenged mice exerted similar suppressor activity in vitro, GITR triggering abrogated GITR+/+ Treg suppressor activity and costimulated CD4+CD25- GITR+/+ effector cells. Furthermore, Treg cells from GITR-/- protected more than Treg cells from GITR+/+ mice against CIA when cotransferred with Treg-depleted splenocytes from arthritic GITR+/+ animals into severe combined immunodeficient (SCID) mice. In conclusion, GITR plays a critical role in the immunological response against CII and in the development of CIA.

Background: Synovitis, which is characterized by infiltration of inflammatory cells, often accompanies progression of clinical symptoms of the temporomandibular joint (TMJ). Synovial fibroblasts of the TMJ are believed to play important roles in progression of synovitis. The purpose of this study was to examine production and gene expression of chemokines by synovial fibroblasts stimulated by tumor necrosis factor-alpha (TNF-alpha)., Methods: Protein levels of chemokines were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression of chemokines was analyzed by real-time polymerase chain reaction (PCR)., Results: Production of interleukin (IL)-8, growth-related oncogene (GRO)-alpha, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T-cell expressed and secreted (RANTES) protein by synovial fibroblasts was increased by TNF-alpha. In contrast, stromal cell-derived factor (SDF)-1alpha, macrophage inflammatory protein (MIP)-1alpha and -1beta were not detectable in conditioned media of synovial fibroblasts, with or without TNF-alpha treatment. Increases in gene expression of IL-8, GRO-alpha, MCP-1, and RANTES in response to TNF-alpha treatment were detected., Conclusions: Increased protein production and gene expression of chemokines by synovial fibroblasts in response to TNF-alpha treatment appears to play an important role in recruitment of inflammatory cells into synovium and the progression of synovitis in the TMJ.

Background: Genes and mechanisms of action involved in human acute rejection after allogeneic heart transplantation remain to be elucidated. The use of a murine allograft model in tandem with cDNA arrays and quantitative real-time polymerase chain reaction (Q-PCR) can greatly help in identifying key genes implicated in human heart acute rejection., Methods and Results: Hearts from Balb/c mice were either not transplanted or transplanted heterotopically in the abdomen of Balb/c (isografts) and C57BL/6 (allografts) mice. Histological analysis showed acute rejection only in allografts. Total RNA was extracted from isografts (n=3), allografts (n=4), and not transplanted hearts (n=4); reverse transcribed; and labeled with P32. Each probe was hybridized to cDNA macroarrays. Eight genes were overexpressed and 7 genes were underexpressed in allografts compared with isografts. Macrophage inflammatory protein-1beta (MIP-1beta), an overexpressed gene, and VE-cadherin, an underexpressed gene, were validated by immunohistochemistry and Q-PCR in the murine models. Genes of interest, validated in the 3 murine groups, were then investigated in human heart tissues. Immunohistochemistry and Q-PCR performed on endomyocardial biopsies after heart transplantation showing no rejection (n=10) or grade IB (n=10) or IIIA (n=10) rejection, according to International Society of Heart and Lung Transplantation criteria, confirmed the results obtained from the murine model., Conclusions: We have demonstrated that the upregulation of MIP-1beta and downregulation of VE-cadherin may strongly participate in human acute heart rejection.

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.

The pathogenesis of myocarditis during Trypanosoma cruzi infection is poorly understood. We investigated the role played by chemokine receptor 5 (CCR5) in the influx of T cells to the cardiac tissue of T. cruzi-infected mice. mRNA and protein for the CCR5 ligands CCL3, CCL4, and CCL5 were detected in the hearts of infected mice in association with CD4+ and CD8+ T cells. There was a high level of CCR5 expression on CD8+ T cells in the hearts of infected mice. Moreover, CCR5 expression on CD8+ T cells was positively modulated by T. cruzi infection. CCR5-deficient mice infected with T. cruzi experienced a dramatically inhibited migration of T cells to the heart and were also more susceptible to infection. These results suggest that CCR5 and its ligands play a central role in the control of T cell influx in T. cruzi-infected mice. Knowledge of the mechanisms that trigger and control the migration of cells to the heart in patients with Chagas disease may help in the design of drugs that prevent myocarditis and protect against the development of severe disease.