OBJECTIVE

Patients with serious traumatic injury and major burns and an animal model of burn injury were studied to determine the effect of injury on the production of cytokines typical of the T helper-2 lymphocyte phenotype as opposed to the T helper-1 phenotype and on the production of interleukin-12.

BACKGROUND

Perturbations of natural and adoptive immunity are related to the increased susceptibility to infection manifested by seriously injured and burn patients. Earlier work has shown that impaired adoptive immunity after injury is characterized by diminished production of interleukin-2 (IL-2), a product of Th lymphocytes. Exposure of naive Th cells to certain antigens and cytokines causes conversion to either the Th-1 or the Th-2 phenotype. Th-1 cells produce IL-2 and interferon-gamma (IFN-tau) and initiate cellular immunity. Th-2 cells secrete interleukin-4 (IL-4) and interleukin-10 (IL-10) and stimulate production of certain antibodies. Conversion to the Th-1 phenotype is facilitated by IL-12, and conversion to the Th-2 phenotype is promoted by IL-4. The authors believed that serious injury might cause conversion of Th cells to the Th-2 as opposed to the Th-1 phenotype rather than generalized Th suppression.

METHODS

The authors studied circulating peripheral blood mononuclear cells (PBMC) from 16 major burn and 8 trauma patients on 32 occasions early after injury and from 13 age- and sex-matched healthy individuals for cytokine production after phytohemagglutinin stimulation. Also studied was a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from burn mice, 10 to 12 per group, were studied after activation by concanavalin A or by the bacterial antigen Staphylococcus aureus Cowan strain I for cytokine production and cytokine messenger RNA expression as determined by reverse transcriptase polymerase chain reaction. Burn mice were compared with sham-burn controls and attention was focused on day 10 after burn injury, a time when IL-2 production and resistance to infection are highly suppressed. Finally, burn and sham-burn animals, 20 per group, were treated in vivo with IL-12 (25 ng daily for 5 days) and observed for mortality after septic challenge (cecal ligation and puncture [CLP]) performed on day 10 after injury.

RESULTS

Peripheral blood mononuclear cells from burn and trauma patients produced less IFN-tau, the index cytokine of Th-1 cells, than PBMCs from healthy individuals 1 to 14 days after burn injury (SE = 77.6 +/- 16 pg/mL patients vs. 141.3 +/- 35 pg/mL controls, p < 0.05). However, production of IL-4, the index cytokine of Th-2 cells, by patient PBMCs was increased (51.0 +/- 13.0 pg/mL patients vs. 26.9 +/- 2.5 controls, p < 0.05). Splenocytes from mice 10 days after burn injury, when compared with sham-burn controls, showed diminished production of IL-2 (1.04 +/- 0.91 units/mL burns vs. 5.8 +/- 0.55 units/mL controls, p < 0.05) and IFN-tau (1.05 +/- 0.7 units/mL burns vs. 12.0 +/- 8.9 units/mL controls, p < 0.05). However, burn splenocytes produced more IL-4 (2492 +/- 157.0 pg/mL burns vs. 672.0 +/- 22.7 pg/mL controls, p < 0.01) and IL-10 (695.2 +/- 20.8 pg/mL burns vs. 567.0 +/- 16.7 pg/mL controls, p < 0.05). Splenocyte production of IL-12 was also reduced after burn (0.20 +/- 0.035 units/mL) as compared with sham burn (0.46 +/- 0.08 units/mL, p < 0.05). The reduction in IL-2, IFN-tau, and IL-12 production by burn splenocytes was reflected by a tenfold decrease in expression of their respective cytokine mRNAs. In vivo IL-12 treatment of burn animals decreased mortality from CLP on day 10 after injury from 85% to 15% (sham-burn mortality after CLP, 15%, p < 0.05) and increased splenocyte IFN-tau production to supranormal levels.

CONCLUSIONS

Serious injury induced diminished production of IL-1 2 and a shift to the Th-2 phenotype with increased production of IL-4 and IL-10, cytokines known to inhibit Th-1 function. The ability of exogenous IL-12 to restore Th-1 cytokine production and resistance to infection suggests a therapeutic role for IL-12 in the immune dysfunction seen after major injury.

Although 20%-40% of persons with acute hepatitis C virus (HCV) infection demonstrate spontaneous clearance, the time course and factors associated with clearance remain poorly understood. We investigated the time to spontaneous clearance and predictors among participants with acute HCV using Cox proportional hazards analyses. Data for this analysis were drawn from an international collaboration of nine prospective cohorts evaluating outcomes after acute HCV infection. Among 632 participants with acute HCV, <em>35</em>% were female, 82% were Caucasian, 49% had <em>interleukin</em>-28 (IL28)B CC genotype (rs12979860), 96% had injected drugs ever, 47% were infected with HCV genotype 1, and 7% had human immunodeficiency virus (HIV) coinfection. Twenty-eight percent were HCV antibody negative/RNA positive at the time of acute HCV detection (early acute HCV). During follow-up, spontaneous clearance occurred in 173 of 632, and at 1 year after infection, 25% (95% confidence interval [CI]: 21, 29) had cleared virus. Among those with clearance, the median time to clearance was 16.5 weeks (IQR: 10.5, 33.4), with 34%, 67%, and 83% demonstrating clearance at 3, 6, and 12 months. Adjusting for age, factors independently associated with time to spontaneous clearance included female sex (adjusted hazards ratio [AHR]: 2.16; 95% CI: 1.48, 3.18), IL28B CC genotype (versus CT/TT; AHR, 2.26; 95% CI: 1.52, 3.34), and HCV genotype 1 (versus non-genotype 1; AHR: 1.56; 95% CI: 1.06, 2.30). The effect of IL28B genotype and HCV genotype on spontaneous clearance was greater among females, compared to males.

CONCLUSIONS

Female sex, favorable IL28B genotype, and HCV genotype 1 are independent predictors of spontaneous clearance. Further research is required to elucidate the observed sex-based differences in HCV control.

Inflammation is thought to play a central role in the etiology and outcome of atherosclerosis. Animal studies as well as in vitro and in vivo human studies suggest that host factors modulate the magnitude and extent of inflammatory responses. We investigated familial aggregation of three systemic markers of inflammation (C-reactive protein (CRP), white blood cell count (WBC), and albumin) in a large, cross-sectional study conducted in four US communities. We found evidence of substantial heritability (<em>35</em>-40%) for CRP levels as well as for WBC and albumin levels. Negligible spouse correlations suggested little influence of shared household environment on these traits. The combination of sociodemographic factors (age, center, education), behavioral and lifestyle factors (cigarette smoking, alcohol intake, hormone replacement therapy), obesity and fat patterning, and prevalent diabetes explained 13-30% the interindividual variability of these traits. There was no evidence that these inflammation phenotypes were linked to a microsatellite marker in the <em>interleukin</em>-1 gene cluster on chromosome 2q, a region that includes several candidate genes for chronic inflammatory diseases. Our findings suggest that CRP levels, albumin levels, and WBC are determined at least partially by genetic factors. Further efforts to identify gene loci affecting these traits are warranted.

The role of glucocorticoids in the treatment of chronic obstructive pulmonary disease (COPD) is controversial. We have previously described high numbers of neutrophils and high concentrations of the inflammatory cytokines <em>interleukin</em>-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha), and of the cell activation markers eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), myeloperoxidase (MPO), and human neutrophil lipocalin (HNL) in COPD patients as compared with controls, and have postulated that the cytokines TNF-alpha and IL-8 play a role in propagating the inflammatory response in COPD. We have now studied the effects of inhaled and oral glucocorticoids on these inflammatory indices in induced sputum. Initially, we studied the effect of a 2-wk course of inhaled budesonide (800 mg twice daily for 2 wk) in 13 patients with severe COPD (mean FDV1: <em>35</em>% predicted). There was no clinical benefit in either lung function or symptom scores, and no significant change in the inflammatory indices as measured by total and differential cell counts and concentrations of TNF-alpha eosinophil activation markers ECP and EPO, and neutrophil activation markers MPO and HNL. Because the lack of anti-inflammatory effect might have been due to poor drug delivery as a result of severe airflow limitation, we undertook a study examining the antiinflammatory effect of oral prednisolone (30 mg daily for 2 wk) in patients with COPD and undertook the same measurements in 10 patients with atopic asthma. Sputum eosinophil numbers, ECP, and EPO were significantly reduced in the asthmatic patients but were not modified in COPD. This confirms the clinical impression that inhaled steroids have little antiinflammatory effect, at least in the short term in this group of patients, and suggests that the inflammatory process in COPD is resistant to the antiinflammatory effect of glucocorticoids.

BACKGROUND

Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models.

METHODS

Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided.

RESULTS

Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P < .001) and 38% (95% CI = 28% to 48%; P < .001) in the HeyA8 and SKOV3ip1 models, respectively.

CONCLUSIONS

Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.

Intrapulmonary activation of leukocytes and release of cellular mediators and enzymes are involved in the pathophysiology of the adult respiratory distress syndrome (ARDS). To investigate a possible role of local cytokines, we measured bronchoalveolar fluid (BALF) and plasma levels of tumor necrosis factor alpha (TNF-alpha) and its soluble inhibitors (sTNF-RI + RII), <em>interleukin</em>-1 beta (IL-1 beta), interferon-alpha (IFN-alpha), and granulocyte elastase in 14 patients at risk for ARDS and in <em>35</em> patients developing ARDS after trauma, sepsis, or shock. During clinical development of severe ARDS, BALF cytokines increased markedly: TNF-alpha from 116 +/- 36 to 10,731 +/- 5,048 pg/ml (mean +/- SEM), p = 0.001; sTNF-RI + RII from 3.7 +/- 1.4 to 24.6 +/- 2.6 ng/ml, p less than 0.05; and IL-1 beta from 7,746 +/- 5,551 to 42,255 +/- 19,176 pg/ml, p = 0.01. Plasma cytokines were not increased in most patients, nor were they correlated with the development or severity of ARDS. BALF elastase was higher in patients developing ARDS than in those at risk but not going into pulmonary failure (0.97 +/- 0.26 versus 0.28 +/- 0.13 U/ml, p = 0.026), and the highest values were observed in the early stages of severe ARDS (1.85 +/- 0.39 U/ml). BALF elastase levels correlated with IFN-alpha (r = 0.72, p less than 0.001). In conclusion, local release of TNF-alpha and IL-1 beta, possibly by pulmonary macrophages or other cells, and/or accumulation in the lung is associated with the development of ARDS.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Chemotherapy is the standard of care for advanced stages of non-small-cell lung cancer (NSCLC). TG4010 is a targeted immunotherapy based on a poxvirus (modified vaccinia virus Ankara) that codes for MUC1 tumour-associated antigen and interleukin 2. This study assessed TG4010 in combination with first-line chemotherapy in advanced NSCLC.

METHODS

148 patients with advanced (stage IIIB [wet] or IV) NSCLC expressing MUC1 by immunohistochemistry, and with performance status 0 or 1, were enrolled in parallel groups in this open-label, phase 2B study. 74 patients were allocated to the combination therapy group, and received TG4010 (10(8) plaque forming units) plus cisplatin (75 mg/m(2) on day 1) and gemcitabine (1250 mg/m(2) on days 1 and 8) repeated every 3 weeks for up to six cycles. 74 patients allocated to the control group received the same chemotherapy alone. Patients were allocated using a dynamic minimisation procedure stratified by centre, performance status, and disease stage. The primary endpoint was 6-month progression-free survival (PFS), with a target rate of 40% or higher in the experimental group. Analyses were done on an intention-to-treat basis. This study is completed and is registered with ClinicalTrials.gov, number NCT00415818.

RESULTS

6-month PFS was 43·2% (32 of 74; 95% CI 33·4-53·5) in the TG4010 plus chemotherapy group, and 35·1% (26 of 74; 25·9-45·3) in the chemotherapy alone group. Fever, abdominal pain, and injection-site pain of any grade according to National Cancer Institute Common Toxicity Criteria were more common in the TG4010 group than in the chemotherapy alone group: 17 of 73 patients (23·3%) versus six of 72 (8·3%), 12 (16·4%) versus two (2·8%), and four (5·5%) versus zero (0%), respectively. The most common grade 3-4 adverse events were neutropenia (33 [45·2%] of patients in the TG4010 plus chemotherapy group vs 31 [43·1%] in the chemotherapy alone group) and fatigue (18 [24·7%] vs 13 [18·1%]); the only grade 3-4 events that differed significantly between groups were anorexia (three [4·1%] vs 10 [13·9%]) and pleural effusion (none vs four [5·6%]). 38 of 73 patients (52·1%) in the TG4010 plus chemotherapy group and 34 of 72 (47·2%) in the chemotherapy alone group had at least one serious adverse event.

CONCLUSIONS

This phase 2B study suggests that TG4010 enhances the effect of chemotherapy in advanced NSCLC. A confirmatory phase 2B-3 trial has been initiated.

BACKGROUND

Transgene SA, Advanced Diagnostics for New Therapeutic Approaches (ADNA)/OSEO.

Eukaryotic messenger RNAs are translated with unequal efficiencies in vivo and in vitro and the molecular basis of this phenomenon is not understood. As an approach to understanding the role of the 5' untranslated leader sequence in regulating mRNA translational efficiency, chimaeric mRNAs have been generated by joining a heterologous leader to complementary DNA (cDNA) sequences, followed by in vitro transcription using SP6 RNA polymerase and in vitro protein synthesis. We used the untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), a well-translated, highly competitive message, to replace the leader sequence of barley alpha-amylase (B alpha A) and human <em>interleukin</em> 1 beta (IL-1 beta) cDNAs. Deletion of transcribed vector sequences and replacement of the native untranslated region with the AMV RNA 4 leader can result in as much as a <em>35</em>-fold increase in mRNA translational efficiency; moreover, the translational efficiency of the chimaeric mRNAs containing the AMV RNA 4 leader is at least as great as that of virion RNA 4. The results suggest that the chimaeric AMV-mRNAs have either a higher relative affinity or a diminished requirement for a limiting component(s) of the translational machinery; in addition, it may be feasible, through use of heterologous leader sequences, to increase expression of engineered genes or cDNAs without changing the antigenic or biological properties of the encoded protein.

OBJECTIVE

To evaluate the interaction between tumor-infiltrating macrophages and cancer cells and its effect on the expression of a potent angiogenic factor, interleukin-8 (IL-8), tumor angiogenesis, and patient outcome in non-small cell lung cancer (NSCLC).

METHODS

We measured tumor IL-8 mRNA expression (by real-time quantitative reverse transcription-PCR), intratumor microvessel counts, and tumor-infiltrating macrophage density (by immunohistochemical staining) in 35 NSCLC surgical specimens and correlated with the patient's clinical outcome. We then investigated the interaction between macrophages (cell line THP-1) and six different human cancer cell lines (four NSCLCs, one osteosarcoma, and one hepatoma) and its effect on IL-8 mRNA expression using a macrophage/cancer cell coculture system, IL-8 mRNA expression in lung cancer cells, and macrophages being measured separately after coculture in the presence or absence of six anti-inflammatory agents, i.e., pentoxifylline, aspirin, indomethacin, dexamethasone, celecoxib (a selective cyclooxygenase-2 inhibitor), and pyrrolidine dithiocarbamate, a specific nuclear factor kappaB (NF-kappaB) inhibitor. NF-kappaB transcriptional activity and protein levels were measured by reporter gene assay and Western blot.

RESULTS

The tumor-infiltrating macrophage density correlated significantly and positively with tumor IL-8 mRNA expression and intratumor microvessel counts and significantly and negatively with patient survival. In addition, after cell-cell interaction in cancer cell:macrophage cocultures, marked IL-8 mRNA expression was induced in lung cancer cells (approximately 270-fold) and, to a lesser degree, in macrophages (4.5-fold). The increase in IL-8 mRNA expression correlated with the in vitro metastatic potential of the cancer cells. All six anti-inflammatory agents suppressed induction of IL-8 mRNA expression in lung cancer cells by >90%, four (pentoxifylline, celecoxib, pyrrolidine dithiocarbamate, and dexamethasone) having a dose-dependent effect. NF-kappaB transcriptional regulation and protein levels were simultaneously increased in the nuclei of cancer cells in macrophage/cancer cell cocultures, this effect also being suppressed by all six anti-inflammatory agents.

CONCLUSIONS

The interaction between infiltrating macrophages and cancer cells up-regulates IL-8 mRNA expression, especially in the cancer cells; this may contribute greatly to the increased tumor angiogenesis and adverse outcome in NSCLC patients with a high density of tumor-infiltrating macrophages. Anti-inflammatory agents can suppress the induction of IL-8 mRNA expression seen in lung cancer cells after coculture with macrophages, and this suppression is mediated, in part, through the NF-kappaB pathway.

<em>Interleukin</em>-6 (IL-6) is a neurotrophic cytokine expressed in both neurons and glia. The present study shows that cerebral ischemia produced by permanent occlusion of the middle cerebral artery (MCAO) produces a dramatic increase in IL-6 bioactivity in the ischemic hemisphere within 2 hours of MCAO (167 +/- 55 IU versus sham: 50 +/- <em>35</em> IU), with further increases at 8 hours (3,456 +/- 1,162 IU) and 24 hours (6,088 +/- 1,772 IU). In a separate series of experiments, intracerebroventricular injection of recombinant IL-6 (3,100 or 31,000 IU) significantly reduced ischemic brain damage after MCAO (to 52% and 65% of controls, respectively). The large increase in endogenous IL-6 bioactivity in response to ischemia, together with the marked neuroprotection produced by exogenous IL-6 suggest that this cytokine is an important endogenous inhibitor of neuronal death during cerebral ischemia.

OBJECTIVE

To investigate the performance of classic risk factors, and of some new biomarkers, in predicting cardiovascular mortality in very old people from the general population with no history of cardiovascular disease.

METHODS

The Leiden 85-plus Study (1997-2004) is an observational prospective cohort study with 5 years of follow-up.

METHODS

General population of the city of Leiden, the Netherlands.

METHODS

Population based sample of participants aged 85 years (215 women and 87 men) with no history of cardiovascular disease; no other exclusion criteria. Main measurements Cause specific mortality was registered during follow-up. All classic risk factors included in the Framingham risk score (sex, systolic blood pressure, total and high density lipoprotein cholesterol, diabetes mellitus, smoking and electrocardiogram based left ventricular hypertrophy), as well as plasma concentrations of the new biomarkers homocysteine, folic acid, C reactive protein, and interleukin 6, were assessed at baseline.

RESULTS

During follow-up, 108 of the 302 participants died; 32% (35/108) of deaths were from cardiovascular causes. Classic risk factors did not predict cardiovascular mortality when used in the Framingham risk score (area under receiver operating characteristic curve 0.53, 95% confidence interval 0.42 to 0.63) or in a newly calibrated model (0.53, 0.43 to 0.64). Of the new biomarkers studied, homocysteine had most predictive power (0.65, 0.55 to 0.75). Entering any additional risk factor or combination of factors into the homocysteine prediction model did not increase its discriminative power.

CONCLUSIONS

In very old people from the general population with no history of cardiovascular disease, concentrations of homocysteine alone can accurately identify those at high risk of cardiovascular mortality, whereas classic risk factors included in the Framingham risk score do not. These preliminary findings warrant validation in a separate cohort.

The distribution of specific toll-like receptors and components of the signaling pathways activated by Pseudomonas aeruginosa flagella were studied in airway epithelial cells. Initially flagella bound to the apical surface of polarized epithelial cells, where they prominently colocalized with asialoGM1. By 4 h of exposure to flagella, toll-like receptor (TLR)5 expression was induced, mobilized to the apical surface of the cells, and colocalized with superficial flagella. <em>Interleukin</em>-8 expression in airway cells was activated by flagella through induction of Ca(2+) fluxes, Src, Ras, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and nuclear factor-kappaB activation, a pathway previously associated with asialoGM1-mediated stimuli. There was evidence for participation of asialoGM1 and TLR2 as well as TLR5 in the response to flagella, and increased asialoGM1 correlated directly with increased signaling. TLR2 DN or TLR5 DN mutations inhibited <em>interleukin</em>-8 induction by 78% and <em>35</em>%, respectively (P < 0.001 for each). The participation of TLR2 as well as TLR5 was confirmed in Chinese hamster ovary cells transfected with either human TLR2 or TLR5 in which flagella activated a nuclear factor-kappaB-luciferase reporter to the same extent. Flagella signaling in airway cells can be initiated by interactions with asialoGM1 and TLR2 as well as by activation of TLR5. The availability of exposed receptors on the apical surface of polarized airway epithelial cells is a major factor in the activation of signaling pathways by flagella.

Periostin is an extracellular matrix protein that has been primarily studied in the context of the heart, where it has been shown to promote cardiac repair and remodeling. In this study, we focused on the role of periostin in an allergic eosinophilic inflammatory disease (eosinophilic esophagitis (EE)) known to involve extensive tissue remodeling. Periostin was indeed markedly overexpressed (<em>35</em>-fold) in the esophagus of EE patients, particularly in the papillae, compared with control individuals. Periostin expression was downstream from transforming growth factor-beta and <em>interleukin</em>-13, as these cytokines were elevated in EE esophageal samples and markedly induced periostin production by primary esophageal fibroblasts (107- and 295-fold, respectively, at 10 ng ml(-1)). A functional role for periostin in eliciting esophageal eosinophilia was demonstrated, as periostin-null mice had a specific defect in allergen-induced eosinophil recruitment to the lungs and esophagus (66 and 72% decrease, respectively). Mechanistic analyses revealed that periostin increased (5.8-fold) eosinophil adhesion to fibronectin. As such, these findings extend the involvement of periostin to esophagitis and uncover a novel role for periostin in directly regulating leukocyte (eosinophil) accumulation in T helper type 2-associated mucosal inflammation in both mice and humans.

Chronic obstructive pulmonary disease (COPD) exacerbations are associated with increased airway and systemic inflammation, though relationships between exacerbation recovery, recurrent exacerbation and inflammation have not been previously reported. In the present study, inflammatory changes at COPD exacerbations were related to clinical nonrecovery and recurrent exacerbations within 50 days. Serum <em>interleukin</em> (IL)-6, C-reactive protein (CRP), sputum IL-6 and IL-8 were measured in 73 COPD patients when stable, at exacerbation and at 7, 14 and <em>35</em> days post-exacerbation. In 23% of patients, symptoms did not recover to baseline by day <em>35</em>. These patients had persistently higher levels of serum CRP during the recovery period. A total of 22% of the patients who had recurrent exacerbations within 50 days had significantly higher levels of serum CRP at day 14, compared with those without recurrences: 8.8 mg.L(-1) versus 3.4 mg.L(-1). Frequent exacerbators had a smaller reduction in systemic inflammation between exacerbation onset and day <em>35</em> compared with infrequent exacerbators. Nonrecovery of symptoms at chronic obstructive pulmonary disease exacerbation is associated with persistently heightened systemic inflammation. The time course of systemic inflammation following exacerbation is different between frequent and infrequent exacerbators. A high serum C-reactive protein concentration 14 days after an index exacerbation may be used as a predictor of recurrent exacerbations within 50 days.

<em>Interleukin</em>-1 (IL-1), the main cytokine instigator of cartilage degeneration in arthritis, induces matrix metalloproteinase-3 (MMP-3) and MMP-13 RNA and protein in chondrocytes. The molecular mechanisms of this induction were investigated with specific inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and activating protein (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors. IL-1 rapidly induced the activation of extracellular-signal regulated kinase (ERK), protein 38 (p38) and c-Jun N-terminal kinase (JNK) MAPKs in the first-passage human femoral head OA chondrocytes. The ERK-MAPK pathway inhibitor, PD98059, attained 46-53% (MMP-3) and 59-66% (MMP-13) inhibition of RNA induction in human OA and 47-52% (MMP-3) and 69-73% (MMP-13) inhibition in bovine chondrocytes. U0126 conferred 37-77% (MMP-3) and 43-73% (MMP-13) suppression in human and 77-100% (MMP-3) and 96-100% (MMP-13) in bovine chondrocytes. P38 and JNK inhibitor, SB20<em>35</em>80 caused <em>35</em>-37% reduction of MMP-3 and MMP-13 RNA in human and 36-46% (MMP-3) and 60-88% (MMP-13) in bovine chondrocytes. Inhibitor of JNK, AP-1 and NF-kappa B, curcumin, achieved 48-99% suppression of MMP-3 and 45-97% of MMP-13 in human and 8-100% (MMP-3) and 32-100% (MMP-13) in bovine chondrocytes. NF-kappaB inhibitor, pyrrolidine dithiocarbamate yielded 83-84% reduction of MMP-3 and 38-55% for MMP-13 in human chondrocytes. In bovine chondrocytes, the induction decreased by 54-64% for MMP-3 and 74-93% for MMP-13 RNA. These results suggest the involvement of MAPKs, AP-1 and NF-kappa B transcription factors in the IL-1 induction of MMPs in chondrocytes. Inhibition of IL-1 signal transduction by these agents could be useful for reducing cartilage resorption by MMPs in arthritis.

OBJECTIVE

The purpose of this study was to determine whether funisitis (inflammation of the umbilical cord detected by histologic examination of the placenta) is associated with changes in the umbilical cord plasma concentration of interleukin 6, microbial invasion of the amniotic cavity, and neonatal sepsis.

METHODS

The relationship among the presence of funisitis, interleukin 6 concentrations in umbilical cord plasma at birth, the results of amniotic fluid culture performed within 3 days of birth, and the occurrence of congenital neonatal sepsis was examined in 315 consecutive singleton preterm births (20-35 weeks' gestation). Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton jelly. The interleukin 6 concentration was measured with a specific immunoassay. Amniocentesis was performed in 106 patients within 3 days of birth. Amniotic fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas.

RESULTS

(1) Funisitis was present in 25% of patients (78/315). (2) Patients with funisitis had a significantly higher median cord plasma interleukin 6 and a lower gestational age at birth than did those without funisitis (cord interleukin 6: median, 52.4 pg/mL; range, 0.9-19,230 pg/mL; vs median, 4.6 pg/mL; range, 0-18,108 pg/mL; gestational age: median, 31.1 weeks' gestation; range, 21.0-35.0 weeks' gestation; vs median, 32.9 weeks' gestation; range, 21.4-35.0 weeks' gestation; P<.001 for each comparison). (3) A cord plasma interleukin 6 of>> or =17.5 pg/mL had a sensitivity of 70% and a specificity of 78% in the identification of funisitis. (4) Microbial invasion of the amniotic cavity and clinical chorioamnionitis were more common among patients with funisitis than among those without funisitis (positive amniotic fluid culture: 53% [20/38]; vs. 12% [8/68]; clinical chorioamnionitis: 18% [14/78]; vs. 4% [9/237]; P<.001 for each comparison). (5) Neonates with funisitis had a significantly higher rate of congenital sepsis than did those without this lesion (12% [8/66] vs. 1% [3/216]; P<.001); this difference remained significant after adjustment for gestational age at birth (odds ratio, 7.2; 95% confidence interval, 1.8-29.0).

CONCLUSIONS

(1) Umbilical cord plasma interleukin 6 concentrations were higher in neonates born with funisitis than in those without this lesion. (2) Funisitis is associated with amniotic fluid infection, congenital neonatal sepsis, and the fetal inflammatory response syndrome.

BACKGROUND

The concentration of C-reactive protein (CRP) in otherwise healthy subjects has been shown to predict future risk of myocardial infarction and stroke. CRP is synthesized by the liver in response to interleukin-6, the serum concentration of which is subject to diurnal variation.

METHODS

To examine the existence of a time-of-day effect for baseline CRP values, we determined CRP concentrations in hourly blood samples drawn from healthy subjects (10 males, 3 females; age range, 21-35 years) during a baseline day in a controlled environment (8 h of nighttime sleep).

RESULTS

Overall CRP concentrations were low, with only three subjects having CRP concentrations >2 mg/L. Comparison of raw data showed stability of CRP concentrations throughout the 24 h studied. When compared with cutoff values of CRP quintile derived from population-based studies, misclassification of greater than one quintile did not occur as a result of diurnal variation in any of the subjects studied. Nonparametric ANOVA comparing different time points showed no significant differences for both raw and z-transformed data. Analysis for rhythmic diurnal variation using a method fitting a cosine curve to the group data was negative.

CONCLUSIONS

Our data show that baseline CRP concentrations are not subject to time-of-day variation and thus help to explain why CRP concentrations are a better predictor of vascular risk than interleukin-6. Determination of CRP for cardiovascular risk prediction may be performed without concern for diurnal variation.

BACKGROUND

The time course and relationship between circulating and local cytokine concentrations, pancreatic inflammation, and organ dysfunction in acute pancreatitis are largely unknown.

METHODS

In a prospective clinical study, we measured the proinflammatory cytokines interleukin (IL)-1 beta, IL-6 and IL-8, the anti-inflammatory cytokine IL-10, interleukin 1 beta receptor antagonist (IL-1RA), and the soluble IL-2 receptor (sIL-2R), and correlated our findings with organ and systemic complications in acute pancreatitis. In 51 patients with acute pancreatitis admitted within 72 hours after the onset of symptoms, these parameters were measured daily for seven days. In addition, 33 aspirates from ascites and the lesser sac were measured.

RESULTS

Sixteen patients had mild acute pancreatitis (AP) and 35 severe AP (Atlanta classification); 18 patients developed systemic complications requiring treatment. All mediators were increased in AP. sIL-2R, IL-10, and IL-6 were significantly elevated in patients with distant organ failure. An imbalance in IL-1 beta/IL-1RA was found in severe AP and pulmonary failure. Peak serum sIL-2R predicted lethal outcome and IL-1RA was an early marker of severity. IL-6 was the best prognostic parameter for pulmonary failure.

CONCLUSIONS

Our results suggest that local mediator release, with a probable IL-1 beta-IL-1RA imbalance in severe cases, is followed by the systemic appearance of pro- and anti-inflammatory mediators. The pattern of local and systemic mediators in complicated AP suggests a role for systemic lymphocyte activation (triggered by local release of mediators) in distant organ complications in severe AP.

Anabolic hormones, mechanical loading, and the obese protein leptin play separate roles in maintaining bone mass. We have previously shown that leptin, as well as its receptor, are expressed by normal human osteoblasts. Consequently, we have investigated how leptin affects proliferation, differentiation, and apoptosis of human osteoblasts. Iliac crest osteoblasts, incubated with either leptin (100 ng/ml), calcitriol (1,25(OH)(2)D(3); 10(-9) M) or 1-84 human parathyroid hormone (PTH; 10(-8) M), were cultured for <em>35</em> consecutive days and assayed for expression of various differentiation-related marker genes (as estimated by RT-PCR), de novo collagen synthesis, proliferation, in vitro mineralization, and osteoclast signaling. The effects of leptin on protection against retinoic acid (RA; 10(-7) M) induced apoptosis, as well as transition into preosteocytes, were also tested. Leptin exposure enhanced cell proliferation and collagen synthesis over both control condition and PTH exposure. Leptin inhibited in vitro calcified nodule production after 1-2 weeks in culture, however, subsequent to 4-5 weeks, leptin significantly stimulated mineralization. The mineralization profile throughout the entire incubation period was almost undistinguishable from the one induced by PTH. In comparison, 1,25(OH)(2)D(3) generally reduced proliferation and collagen production rates, whereas mineralization was markedly enhanced. Leptin exposure (at 2 and 5 weeks) significantly enhanced the expression of TGFbeta, IGF-I, collagen-Ialpha, ALP, and osteocalcin mRNA. Leptin also protected against RA-induced apoptosis, as estimated by soluble DNA fractions and DNA laddering patterns subsequent to 10 days of culture. The expression profiles of Bax-alpha and Bcl-2 mRNAs indicated that leptin per se significantly protected against apoptosis throughout the entire incubation period. Furthermore, the osteoblast marker OSF-2 was diminished, whereas the CD44 osteocyte marker gene expression was stimulated, indicating a transition into preosteocytes. In terms of osteoclastic signaling, leptin significantly augmented the mRNA levels of both <em>interleukin</em>-6 (IL-6) and osteoprotegerin (OPG). In summary, continuous leptin exposure of iliac crest osteoblasts, promotes collagen synthesis, cell differentiation and in vitro mineralization, as well as cell survival and transition into preosteocytes. Leptin may also facilitate osteoblastic signaling to the osteoclast.

Circulating levels of inflammatory markers can predict cardiovascular disease risk. To identify genes influencing the levels of these markers, we genotyped 1,343 single-nucleotide polymorphisms (SNPs) in 1,184 African Americans from the Health, Aging and Body Composition (Health ABC) Study. Using admixture mapping, we found a significant association of <em>interleukin</em> 6 soluble receptor (IL-6 SR) with European ancestry on chromosome 1 (LOD 4.59), in a region that includes the gene for this receptor (IL-6R). Genotyping 19 SNPs showed that the effect is largely explained by an allele at 4% frequency in West Africans and at <em>35</em>% frequency in European Americans, first described as associated with IL-6 SR in a Japanese cohort. We replicate this association (P(<1.0x10-12) and also demonstrate a new association with circulating levels of a different molecule, IL-6 (P<3.4x10-5). After replication in 1,674 European Americans from Health ABC, the combined result is even more significant: P(<1.0x10-12 for IL-6 SR, and P<2.0x10-9 for IL-6. These results also serve as an important proof of principle, showing that admixture mapping can not only coarsely localize but can also fine map a phenotypically important variant.

OBJECTIVE

Men with the metabolic syndrome (MetS) have low plasma testosterone (T) levels. The aim of this study was to establish whether the normalization of plasma T improves the features of the MetS.

METHODS

A randomized, placebo-controlled, double-blinded, phase III trial of 184 men suffering from both the MetS and hypogonadism.

METHODS

One hundred and eighty-four men, aged <em>35</em>-70, with the MetS and hypogonadism (baseline total T level <12·0 nm or calculated free T level <225 pm.), recruited in the outpatient andrology and urology clinic, Research Center for Endocrinology in Moscow, Russia.

METHODS

Treatment for 30 weeks with either parenteral T undecanoate (n = 113; TU; 1000 mg IM) or placebo (n = 71), administered at baseline, and after 6 and 18 weeks. One hundred and five (92·9%) men receiving TU and 65 (91·5%) receiving placebo completed the trial.

METHODS

Body weight, body mass index (BMI), waist circumference (WC), hip circumference, waist-to-hip ratio, insulin, leptin, glucose, cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, C-reactive protein (CRP), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α).

RESULTS

There were significant decreases in weight, BMI and WC in the TU vs placebo group. Levels of leptin and insulin also decreased, but there were no changes in serum glucose or lipid profile. Of the inflammatory markers, IL-1β, TNF-α and CRP decreased, while IL-6 and IL-10 did not change significantly.

CONCLUSIONS

Thirty weeks of T administration normalizing plasma T in hypogonadal men with the MetS improved some components of the MetS and a number of inflammatory markers.

We investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF164 (rrSCF164) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of "connective tissue-type mast cells" (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF164 induced the proliferation of cloned <em>interleukin</em> 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC. BMCMC maintained in rrSCF164 not only proliferated but also matured. Prior to exposure to rrSCF164, the BMCMC were alcian blue positive, safranin negative, and berberine sulfate negative; had a histamine content of 0.08 +/- 0.02 pg per cell; and incorporated [<em>35S</em>]sulfate into chondroitin sulfates. After 4 wk in rrSCF164, the BMCMC were predominantly safranin positive and berberine sulfate positive, had a histamine content of 2.23 +/- 0.39 pg per cell, and synthesized <em>35S</em>-labeled proteoglycans that included substantial amounts (41-70%) of [<em>35S</em>]heparin. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.

OBJECTIVE

Elevated lipopolysaccharide-binding protein (LBP), a marker of subclinical endotoxemia, may be involved in the pathogenesis of obesity and metabolic risk. We aimed to investigate the association between plasma LBP and metabolic disorders in apparently healthy Chinese.

METHODS

A population-based study including 559 overweight/obese (BMI>>or=24.0 kg/m(2)) and 500 normal-weight (18.0 <or= BMI <24.0 kg/m(2)) subjects aged <em>35</em>-54 years was conducted in Shanghai, China. Fasting plasma glucose, lipid profile, LBP, high-sensitivity C-reactive protein, <em>interleukin</em>-6, high-molecular-weight (HMW) adiponectin, leptin, hepatic enzymes, and body composition were measured. Metabolic syndrome was defined by the updated National Cholesterol Education Program Adult Treatment Panel III criterion for Asian Americans.

RESULTS

LBP levels were significantly higher in overweight/obese individuals than in normal-weight individuals (geometric mean 27.6 [95% CI 25.2-30.3] vs. 10.0 [9.1-11.1] microg/ml; P < 0.001). After multiple adjustments including BMI, the odds ratios were 3.54 (95% CI 2.05-6.09) and 5.53 (95% CI 2.64-11.59) for metabolic syndrome and type 2 diabetes, respectively, comparing the highest with the lowest LBP quartile. Further adjustments for inflammatory markers almost abolished the significant association of LBP with metabolic syndrome but not that with type 2 diabetes, and controlling for adipokines and hepatic enzymes did not substantially alter the results.

CONCLUSIONS

Elevated circulating LBP was associated with obesity, metabolic syndrome, and type 2 diabetes in apparently healthy Chinese. These findings suggested a role of lipopolysaccharide via initiation of innate immune mechanism(s) in metabolic disorders. Prospective studies are needed to confirm these results.

Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [<em>35S</em>]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine <em>interleukin</em>-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.