Comprehensive analysis of vitreous humor chemokines in type 2 diabetic patients with and without diabetic retinopathy.

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.

Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice.

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.

CCL15 overexpression predicts poor prognosis for hepatocellular carcinoma.

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.

Inflammatory potential in relation to the microbial content of settled dust samples collected from moisture-damaged and reference schools: results of HITEA study.

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.

Dialysate cytokine levels do not predict encapsulating peritoneal sclerosis.

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.

Efeito imunomodulador de vesículas de membrana obtidas de macrófagosinfectados por leishmania sobre macrófagos não infectados / Immunomodulatory Effect of membrane vesicles obtained from macrophages Leishmania infection of uninfected macrophages

Vesículas de membrana (VMs) derivadas de macrófagos infectados com microorganismos intracelulares têm capacidade inflamatória. Estas vesículas podem conter antígenos do patógeno, carrear moléculas de MHC II e componentes celulares que podem atuar como PAMPs ou DAMPs induzindo resposta imune. Na infecção por Leishmania, a indução de uma resposta do tipo Th1 é crucial para promoção de proteção contra o parasito. O objetivo do trabalho foi avaliar a capacidade imunomoduladora de VMs derivadas de macrófagos infectados com L. amazonensis sobre a produção de citocinas por outros macrófagos. VMs foram visualizadas por microscopia eletrônica tanto em preparações celulares como no precipitado obtido por sucessivas centrifugações de sobrenadantes de cultivos celulares. Foi observada por citometria de fluxo a presença de marcadores celulares específicos (F4/80 e CD11b) nas VMs, bem como MHC II. O tratamento de macrófagos não infectados com VMs derivadas de macrófagos infectados com L. amazonensis ocasionou aumento consistente da produção de IL-12p70 e IL-1β. Estas vesículas poderiam, portanto, favorecer a modulação da resposta imune em favor do combate ao parasito.

Membrane vesicles (MV) derived macrophages infected with intracellular microbes are proinflammatory. These vesicles contain antigens of the pathogen, carry MHC II molecules and cellular components that can act as PAMPs or DAMPs inducing immune responses. In Leishmania infection the induction of a Th1 response is crucial for the protection against the parasite. The aim of the study was to evaluate whether vesicles derived from macrophages infected with L. amazonensis had the capacity to modulate the response of other macrophages. MV were visualized by electron microscopy in cellular preparations as well in the precipitate obtained by centrifugation of cell supernatants. Flow cytometry revealed the presence of specific cellular markers (F4/80 and CD11b) in the MV, as well as MHC II. Treatment of noninfected macrophages with MV derived from L. amazonensis-infected macrophages consistently caused increased production of IL-12p70 and IL-1β. These vesicles can favor the modulation of the immune response in favor of combating the parasite.

CC-chemokine CCL15 expression and possible implications for the pathogenesis of IgE-related severe asthma.

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.

Assessment of the selected biochemical markers in predicting preterm labour.

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-3ß (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.

Changes of immunomodulatory cytokines associated with omalizumab therapy for severe persistent asthma.

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.

Molecular analysis of aortic intimal hyperplasia caused by Porphyromonas gingivalis infection in mice with endothelial damage.

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.

Identification of the efflux transporter of the fluoroquinolone antibiotic ciprofloxacin in murine macrophages: studies with ciprofloxacin-resistant cells.

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.

Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis.

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.

Local and systemic release of cytokines, nerve growth factor, prostaglandin E2, and substance P in incisional wounds and serum following cesarean delivery.

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.

Delayed accumulation of activated macrophages and inhibition of remyelination after spinal cord injury in an adult rodent model.

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.

A comparison of inflammatory mediators released by basophils of asthmatic and control subjects in response to high-affinity IgE receptor aggregation.

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.

Chemokine redundancy in BOS pathogenesis. A possible role also for the CC chemokines: MIP3-beta, MIP3-alpha, MDC and their specific receptors.

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.

Transcriptional regulation of inflammatory and extracellular matrix-regulating genes in cerebral arteries following experimental subarachnoid hemorrhage in rats. Laboratory investigation.

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.

Longitudinal comparison of chemokines in breastmilk early postpartum among HIV-1-infected and uninfected Kenyan women.

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.

Reduction of gap and adherens junction proteins and intercalated disc structural remodeling in the hearts of mice submitted to severe cecal ligation and puncture sepsis.

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.