OBJECTIVE

To analyze whether inflammatory disease activity plays a substantial role in the loss of bone mass observed in ankylosing spondylitis (AS) patients who have not yet developed ankylosis.

METHODS

A longitudinal cohort study of 34 patients with early AS (duration <10 years) without ankylosis was conducted. The mean followup was 19 months. Loss of bone mass in defined regions of the lumbar spine and femoral neck was analyzed by dual x-ray absorptiometry. Patients were grouped according to biologic parameters of disease activity (erythrocyte sedimentation rate or C-reactive protein level). Group 1 consisted of 14 patients with active disease; group 2 comprised <em>20</em> patients with inactive disease. Serum levels of interleukin-6 (<em>IL</em>-6) and of hormones (sex, thyroid, and calciotropic), vertebral mobility (Schober test), daily physical activity, and treatment administered were recorded every 6 months for all patients.

RESULTS

At the end of the followup period, patients with active AS showed a significant reduction in bone mass in the lumbar spine (mean 1.01 gm/cm2 at study entry versus 0.961 gm/cm2 at followup [P = 0.005]) and femoral neck (0.849 gm/cm2 versus 0.821 gm/cm2 [P = 0.015]), which represented losses of 5% and 3%, respectively. In contrast, no significant reduction in bone mass was observed in patients with inactive AS. As expected, serum IL-6 levels were significantly higher in patients with active AS than in those with inactive disease (mean +/- SD 8.3 +/- 9 pg/ml versus 2.8 +/- 5 pg/ml [P = 0.008]). No significant differences were observed between the 2 groups in any of the other variables analyzed.

CONCLUSIONS

The observation that loss of bone mass in AS occurred only in patients with persistent active disease strongly suggests that inflammatory activity of the disease itself plays a major role in the pathophysiology of the early bone mineral disorders observed in these patients.

Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photooxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2- to 14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40-60% decrease in proteasome activity, a 50-80% decrease in expression of CFH and MCP-1, and an~<em>20</em>-fold increase in expression of <em>IL</em>-8. The photooxidation-induced changes in expression of MCP-1, <em>IL</em>-8, and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photooxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photooxidation-induced inactivation of the proteasome and photooxidation-induced changes in expression of MCP-1, <em>IL</em>-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photooxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.

BACKGROUND

Neonatal sepsis is a common and life-threatening disorder, particularly among preterm infants. Early initiation of antibiotic therapy is frequently delayed because the first clinical signs of sepsis are non-specific and there are no reliable early laboratory indicators. We investigated the time course of expression and the prognostic power of the early inflammatory mediators interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6), and circulating intercellular adhesion molecule-1 (cICAM-1) before clinical diagnosis of sepsis.

METHODS

In a prospective multicentre study, we monitored 182 very-low-birthweight infants in six intensive-care units for occurrence of sepsis. During routine or clinically indicated blood sampling, an additional sample was collected for measurement of IL-1ra, IL-6, cICAM-1, and C-reactive protein (CRP). Infants were grouped into those with proven sepsis, no infection, or unclassified. The mean study duration was 34 days. Whenever sepsis occurred, a study period of 10 days was defined: day 0 was the day of clinical diagnosis of sepsis; days -4 to -1 were the 4 days before diagnosis; days +1 to +5 were the 5 days after. We compared the concentrations of the immune mediators during the 10-day study period with group-specific baseline values from before day -4.

RESULTS

101 infants were included in the analysis: 21 with proven sepsis, 20 with no infection, and 60 unclassified. We excluded 57 because of incomplete datasets and 24 who had early-onset sepsis. IL-1ra and IL-6 increased significantly 2 days before diagnosis of sepsis; maximum median increases within the study period were 15-fold for IL-1ra and 12-fold for IL-6. The diagnostic sensitivities of IL-1ra, IL-6, and CRP concentrations on day 0 of diagnosis were 93%, 86%, and 43%, respectively; corresponding values on day -1 were 64%, 57%, and 18%. The specificities of IL-1ra, IL-6, and CRP concentrations were 92%, 83%, and 93%. cICAM-1 had a specificity of only 64%.

CONCLUSIONS

IL-1ra and IL-6 are superior to cICAM-1 and CRP as predictors of sepsis 1 or more days before clinical diagnosis. Ad-hoc measurement of these cytokines could allow earlier initiation of antibiotic therapy with corresponding improvement in outcome in very-low-birthweight infants with sepsis.

Opisthorchis viverrini is a fish-borne trematode endemic in East Asia. Following ingestion, the flukes locate to the biliary tre where chronic infection frequently leads to cholangiocarcinoma (CCA). The mechanisms by which O. viverrini infection culminates in CCA remain unknown. An unexplored aspect is its influence on the host microbiome. In the hamster, infection with this pathogen reliably leads to CCA. Genomic DNAs of microbiota from colorectal contents and bile of hamsters and from whole O. viverrini were examined in this model of fluke-induced CCA. Microbial communities were characterized by high-throughput sequencing of variable regions 7-9 of prokaryotic 16S ribosomal DNA. Of ∼1 million sequences, 536,009 with useable reads were assignable to 29,776 operational taxonomy units (OTUs) and, in turn, to <em>20</em> phyla and 273 genera of Bacteria or Archaea. Microbial community analyses revealed that fluke infection perturbed the gastrointestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae (P≤0.05). More than 60 OTUs were detected in the biliary system, which confirmed bacteriobilia and a noteworthy community of microbes associated with the parasites. The fluke-associated microorganisms included potential pathogens from the Enterobacteriaceae and Listeriaceae and others, including Cyanobacteria and Deinococci, usually found in external environments. Given that opisthorchiasis is distinguished from other helminth infections by a robust inflammatory phenotype with conspicuously elevated <em>IL</em>-6, and that inflammation of the biliary system leads to periductal fibrosis, which is a precursor of CCA, the flukes and their microbiota may together drive this distinctive immune response.

It is unknown whether transforming growth factor beta1 (TGF-beta1) signaling uniformly participates in fibrogenic chronic liver diseases, irrespective of the underlying origin, or if other cytokines such as interleukin (<em>IL</em>)-13 share in fibrogenesis (e.g., due to regulatory effects on type I pro-collagen expression). TGF-beta1 signaling events were scored in 396 liver tissue samples from patients with diverse chronic liver diseases, including hepatitis B virus (HBV), hepatitis C virus (HCV), Schistosoma japonicum infection, and steatosis/steatohepatitis. Phospho-Smad2 staining correlated significantly with fibrotic stage in patients with HBV infection (n = 112, P < 0.001) and steatosis/steatohepatitis (n = 1<em>20</em>, P < 0.01), but not in patients with HCV infection (n = 77, P>> 0.05). In tissue with HBx protein expression, phospho-Smad2 was detectable, suggesting a functional link between viral protein expression and TGF-beta1 signaling. For <em>IL</em>-13, immunostaining correlated with fibrotic stage in patients with HCV infection and steatosis/steatohepatitis. <em>IL</em>-13 protein was more abundant in liver tissue lysates from three HCV patients compared with controls, as were <em>IL</em>-13 serum levels in 68 patients with chronic HCV infection compared with <em>20</em> healthy volunteers (72.87 +/- 26.38 versus 45.41 +/- 3.73, P < 0.001). Immunohistochemistry results suggest that <em>IL</em>-13-mediated liver fibrogenesis may take place in the absence of phospho-signal transducer and activator of transcription protein 6 signaling. In a subgroup of patients with advanced liver fibrosis (stage>> or =3), neither TGF-beta nor <em>IL</em>-13 signaling was detectable.

CONCLUSIONS

Depending on the cause of liver damage, a predominance of TGF-beta or IL-13 signaling is found. TGF-beta1 predominance is detected in HBV-related liver fibrogenesis and IL-13 predominance in chronic HCV infection. In some instances, the underlying fibrogenic mediator remains enigmatic.

OBJECTIVE

This phase I/II study evaluated safety, efficacy, and pharmacokinetics of escalating, multiple doses of siltuximab, a chimeric anti-interleukin (IL)-6 monoclonal antibody derived from a new Chinese hamster ovary (CHO) cell line in patients with advanced/refractory solid tumors.

METHODS

In the phase I dose-escalation cohorts, 20 patients with advanced/refractory solid tumors received siltuximab 2.8 or 5.5 mg/kg every 2 weeks or 11 or 15 mg/kg every 3 weeks intravenously (i.v.). In the phase I expansion (n = 24) and phase II cohorts (n = 40), patients with Kirsten rat sarcoma-2 (KRAS)-mutant tumors, ovarian, pancreatic, or anti-EGF receptor (EGFR) refractory/resistant non-small cell lung cancer (NSCLC), colorectal, or H&N cancer received 15 mg/kg every 3 weeks. The phase II primary efficacy endpoint was complete response, partial response, or stable disease >6 weeks.

RESULTS

Eighty-four patients (35 colorectal, 29 ovarian, 9 pancreatic, and 11 other) received a median of three (range, 1-45) cycles. One dose-limiting toxicity occurred at 5.5 mg/kg. Common grade ≥3 adverse events were hepatic function abnormalities (15%), physical health deterioration (12%), and fatigue (11%). Ten percent of patients had siltuximab-related grade ≥3 adverse events. Neutropenia (4%) was the only possibly related adverse event grade ≥3 reported in >1 patient. Serious adverse events were reported in 42%; most were related to underlying disease. The pharmacokinetic profile of CHO-derived siltuximab appears similar to the previous cell line. No objective responses occurred; 5 of 84 patients had stable disease >6 weeks. Hemoglobin increased ≥1.5 g/dL in 33 of 47 patients. At 11 and 15 mg/kg, completely sustained C-reactive protein suppression was observed.

CONCLUSIONS

Siltuximab monotherapy appears to be well tolerated but without clinical activity in solid tumors, including ovarian and KRAS-mutant cancers. The recommended phase II doses were 11 and 15 mg/kg every 3 weeks.

Interleukin 4 (<em>IL</em>-4) and <em>IL</em>-13 share many biological functions. Both cytokines promote growth of activated human B cells and induce naive human surface immunoglobulin D+ (sIgD+) B cells to produce IgG4 and IgE. Here we show that a mutant form of human <em>IL</em>-4, in which the tyrosine residue at position 124 is replaced by aspartic acid (h<em>IL</em>-4.Y124D), specifically blocks <em>IL</em>-4 and <em>IL</em>-13-induced proliferation of B cells costimulated by anti-CD40 mAbs in a dose-dependent fashion. A mouse mutant <em>IL</em>-4 protein (m<em>IL</em>-4.Y119D), which antagonizes the biological activity of mouse <em>IL</em>-4, was ineffective. In addition, h<em>IL</em>-4.Y124D, at concentrations of up to 40 nM, did not affect <em>IL</em>-2-induced B cell proliferation. h<em>IL</em>-4.Y124D did not have detectable agonistic activity in these B cell proliferation assays. Interestingly, h<em>IL</em>-4.Y124D also strongly inhibited both <em>IL</em>-4 or <em>IL</em>-13-induced IgG4 and IgE synthesis in cultures of peripheral blood mononuclear cells, or highly purified sIgD+ B cells cultured in the presence of anti-CD40 mAbs. <em>IL</em>-4 and <em>IL</em>-13-induced IgE responses were inhibited>> 95% at a approximately 50- or approximately <em>20</em>-fold excess of h<em>IL</em>-4.Y124D, respectively, despite the fact that the <em>IL</em>-4 mutant protein had a weak agonistic activity. This agonistic activity was 1.6 +/- 1.9% (n = 4) of the maximal IgE responses induced by saturating concentrations of <em>IL</em>-4. Taken together, these data indicate that there are commonalities between the <em>IL</em>-4 and <em>IL</em>-13 receptor. In addition, since h<em>IL</em>-4.Y124D inhibited both <em>IL</em>-4 and <em>IL</em>-13-induced IgE synthesis, it is likely that antagonistic mutant <em>IL</em>-4 proteins may have potential clinical use in the treatment of IgE-mediated allergic diseases.

OBJECTIVE

In the classic model of pleurisy there is little evidence about the anti-inflammatory effects of low-level laser therapy (LLLT) as well the dosage characteristics, such as wavelength, total energy, number and pattern of treatment. In this study we investigated the potential effects of LLLT on modulating the pro-inflammatory and anti-inflammatory mediators of acute inflammation in a rat pleurisy model.

METHODS

A sample of 48 female Wistar rats were divided into control and experiential groups. An inflammation was induced by carrageenan (0.2 ml) injected into the pleural cavity. At 1, 2, and 3 hours after induction a continuous wave (<em>20</em> mW) diode laser of the InGaAlP (660 nm) type was used in the four laser groups with different doses and treatment patterns. One group received a single dose of 2.1 J and the other three groups received a total energy of 0.9, 2.1, and 4.2 J. Four hours later the exudate volume, total and differential leukocytes, protein concentration, NO, <em>IL</em>-6, <em>IL</em>-10, TNF-alpha, and MCP-1 were measured from the aspirated liquid.

RESULTS

All the treatment patterns and quantity of energy studied show significant reduction of the exudate volume (P<0.05). Using energy of 0.9 J only NO, IL-6, MCP-1 and IL-10 are significantly reduced (P<0.05). On the other hand, higher energies (2.1 and 4.2 J) significantly reduce all variables independently of the treatment pattern. The neutrophil migration has a straight correlation with the TNF-alpha (r = 0.551) and NO (r = 0.549) concentration.

CONCLUSIONS

LLLT-660 nm induced an anti-inflammatory effect characterized by inhibition of either total or differential leukocyte influx, exudation, total protein, NO, IL-6, MCP-1, IL-10, and TNF-alpha, in a dose-dependent manner. Under these conditions, laser treatment with 2.1 J was more effective than 0.9 and 4.2 J.

OBJECTIVE

We sought to examine the role of the pro-inflammatory cytokine, interleukin-1-beta (IL-1beta), in the process of left ventricular (LV) remodeling in the early phase after myocardial infarction (MI).

BACKGROUND

Studies have shown that pro-inflammatory cytokines are closely related to the progression of LV remodeling after MI.

METHODS

Mice underwent coronary artery ligation, and the time course of LV remodeling was followed up to 20 weeks. The gene expression level of IL-1beta was examined. In a second set of experiments, the mice underwent coronary artery ligation followed by treatment with anti-IL-1beta antibody (100 microg, intravenously), versus control immunoglobulin G (100 microg, intravenously) immediately after the operation.

RESULTS

Rapid hypertrophy of noninfarcted myocardium was observed by four weeks, and interstitial fibrosis progressed steadily up to 20 weeks. Anti-IL-1beta treatment increased the occurrence of ventricular rupture and suppressed collagen accumulation in the infarct-related area. At four and eight weeks after the operation, total heart weight and LV end-diastolic dimension were significantly greater in the anti-IL-1beta-treated mice than in the other groups. In the infarct-related area, collagen accumulation was suppressed, whereas in the noninfarcted area, pro-collagen gene expression levels, particularly type III, were decreased in the anti-IL-1beta-treated mice.

CONCLUSIONS

Anti-IL-1beta treatment suppressed pro-collagen gene expression and delayed wound healing mechanisms-properties that are likely to lead to progression of LV remodeling. In the acute phase of MI, IL-1beta appears to play a protective role.

WNT5A has recently been implicated in inflammatory processes, but its role as a bone marrow stromal cell (BMSC)-derived mediator of joint inflammation in arthritis is unclear. Here, we investigated whether inflammatory stimuli induce WNT5A in BMSC to control inflammatory responses. WNT5A levels were determined in human BMSC after stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF-α,) and in synovial cells and tissue of patients with rheumatoid arthritis (RA) and human TNF-α transgenic (hTNFtg) mice. A microarray analysis of WNT5A-treated murine osteoblasts was performed using Affymetrix gene chips. The regulation of cytokine/chemokine expression was confirmed by qPCR, ELISA, and Luminex technology in BMSC after stimulation with WNT5A or WNT5A knockdown. Relevant signaling pathways were identified using specific inhibitors. Migration of MACS-purified T lymphocytes and monocytes was assessed using the FluoroBlok system. WNT5A expression was increased threefold in BMSC after stimulation with LPS or TNF-α. Synovial fibroblasts from patients with RA showed a twofold increase of WNT5A expression compared with control cells, and its expression was highly induced in the synovial tissue of patients with RA and hTNFtg mice. Microarray analysis of WNT5A-treated osteoblasts identified cytokines and chemokines as targets. The induction of <em>IL</em>-1β, <em>IL</em>-6, CCL2, CCL5, CXCL1, and CXCL5 by WNT5A was confirmed in BMSC and depended on the activation of the NF-κB, mitogen-activated protein (MAPK), and Akt pathways. Accordingly, knockdown of WNT5A markedly reduced the basal and LPS-induced cytokine/chemokine production. Finally, migration of monocytes and T cells toward the supernatant of WNT5A-treated BMSC was increased by 25% and <em>20</em>%, respectively. This study underlines the critical role of BMSC-derived WNT5A in the regulation of inflammatory processes and suggests its participation in the pathogenesis of RA.

BACKGROUND

Osteoporosis mainly occurs in postmenopausal women, which is characterized by low bone mineral density (BMD) due to unbalanced bone resorption by osteoclasts and formation by osteoblasts. Circulating monocytes play important roles in osteoclastogenesis by acting as osteoclast precursors and secreting osteoclastogenic factors, such as IL-1, IL-6 and TNF-α. MicroRNAs (miRNAs) have been implicated as important biomarkers in various diseases. The present study aimed to find significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.

RESULTS

We used ABI TaqMan® miRNA array followed by qRT-PCR validation in circulating monocytes to identify miRNA biomarkers in 10 high and 10 low BMD postmenopausal Caucasian women. MiR-133a was upregulated (P=0.007) in the low compared with the high BMD groups in the array analyses, which was also validated by qRT-PCR (P=0.044). We performed bioinformatic target gene analysis and found three potential osteoclast-related target genes, CXCL11, CXCR3 and SLC39A1. In addition, we performed Pearson correlation analyses between the expression levels of miR-133a and the three potential target genes in the 20 postmenopausal women. We did find negative correlations between miR-133a and all the three genes though not significant.

CONCLUSIONS

This is the first in vivo miRNA expression analysis in human circulating monocytes to identify novel miRNA biomarkers underlying postmenopausal osteoporosis. Our results suggest that miR-133a in circulating monocytes is a potential biomarker for postmenopausal osteoporosis.

To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = <em>20</em>) induced an up-regulation of ICAM-1, intracellular <em>IL</em>-8, <em>IL</em>-6, <em>IL</em>-15, and surface <em>IL</em>-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-alpha, IFN-gamma, <em>IL</em>-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = <em>20</em>) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = <em>20</em>) were maximal. Blocking Abs to <em>IL</em>-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, <em>IL</em>-17, TNF-alpha, and IFN-gamma significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4- micro m inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of <em>IL</em>-15 induces the proinflammatory cytokines TNF-alpha, IFN-gamma, and <em>IL</em>-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of <em>IL</em>-15, <em>IL</em>-8, and <em>IL</em>-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.

OBJECTIVE

The etiologic role of bacterial pathogens isolated from sputum culture in 40 to 50% of acute exacerbations of chronic bronchitis (AECB) is controversial. If bacterial pathogens cause these AECB, they should be associated with greater neutrophilic airway inflammation than pathogen-negative exacerbations.

METHODS

This hypothesis was tested by comparing levels of interleukin (<em>IL</em>)-8, tumor necrosis factor (TNF)-alpha, and neutrophil elastase (NE) in 81 sputum samples obtained from 45 patients with AECB. Four groups were compared. In the first three groups, nontypable Haemophilus influenzae (n = <em>20</em>), Haemophilus parainfluenzae (n = 27), and Moraxella catarrhalis (n = 14) were isolated as sole pathogens, respectively. In the fourth group, only normal flora was isolated (n = <em>20</em>). Paired samples, obtained from individual patients at different times, that differed in their culture results were also compared.

METHODS

An outpatient research clinic at a Veterans Affairs Medical Center.

METHODS

These patients were participating in a prospective, longitudinal study of the dynamics of bacterial infection in chronic bronchitis, for which they were seen in the study clinic on a monthly basis as well as when they were experiencing symptoms suggestive of AECB.

METHODS

None.

RESULTS

H influenzae exacerbations were associated with significantly higher sputum IL-8, TNF-alpha, and NE. M catarrhalis exacerbations demonstrated significantly higher sputum TNF-alpha and NE when compared to pathogen-negative exacerbations. H parainfluenzae-associated exacerbations had an inflammatory profile similar to pathogen-negative exacerbations. Sputum elastase level distinguished bacterial from nonbacterial AECB and correlated with clinical severity of the AECB.

CONCLUSIONS

Increased airway inflammation associated with isolation of H influenzae and M catarrhalis supports an etiologic role of these pathogens in AECB.

The incidence of cutaneous malignant melanoma in the United States has increased more than any other cancer in recent years. Chemotherapy for metastatic melanoma is disappointing, there being anecdotal cases of complete remission. Dacarbazine (DTIC) is considered the gold standard for treatment, having a response rate of 15-<em>20</em>%, but most responses are not sustained. The mechanisms for the increased chemotherapeutic resistance of melanoma are unclear. The objective of this study was to determine the mechanisms by which melanoma cells escape the cytotoxic effect of DTIC. Here, we show that DTIC induced interleukin (<em>IL</em>)-8 and vascular endothelial growth factor (VEGF) protein overexpression and secretion via transcriptional up-regulation in the two melanoma cell lines SB-2 and MeWo. Luciferase activity driven by the <em>IL</em>-8 and VEGF promoters was up-regulated by 1.5-2- and 1.6-3.5-fold, respectively, in the SB2 and MeWo melanoma cell lines. The mitogen-activated protein kinase signal transduction pathway seemed to regulate at least partially the activation of <em>IL</em>-8, whereas it was not involved in VEGF promoter regulation. Electrophoretic mobility shift analysis analyses have revealed an increase in binding activity of activator protein 1 (c-Jun) and nuclear factor-kappaB after DTIC treatment for both melanoma cell lines. Metastatic melanoma cell lines secreting high levels of <em>IL</em>-8 and VEGF were more resistant to DTIC than early primary melanomas secreting low levels of the cytokines. In addition, transfection of the primary cutaneous melanoma SB-2 cells with the <em>IL</em>-8 gene rendered them resistant to the cytotoxic effect of the drug, whereas the addition of <em>IL</em>-8-neutralizing antibody to metastatic melanoma cells lowered their sensitivity to DTIC. Taken together, our data demonstrate that DTIC can cause melanoma cells to secrete <em>IL</em>-8 and VEGF, which might render them resistant to the cytotoxic effects of the drug. We propose that combination treatment with anti-VEGF/<em>IL</em>-8 agents may potentiate the therapeutic effects of DTIC.

OBJECTIVE

To determine the kinetics of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in serum and aqueous humor of rats with different susceptibilities to endotoxin-induced uveitis (EIU), after footpad injection of lipopolysaccharide (LPS).

METHODS

Samples were collected from EIU-susceptible Lewis rats and EIU-resistant Brown Norway (BN) rats for up to 72 hours after LPS injection. Specific bioassays were used to measure TNF and IL-6 activity. Northern blot analysis was used to assess intraocular IL-6 mRNA expression.

RESULTS

High levels of TNF and IL-6 were detected in serum of both rat strains early after LPS injection. A second rise in serum TNF was observed at 18 to 20 hours in Lewis rats only. In aqueous humor of Lewis rats, high levels of TNF and IL-6 were observed early after LPS injection (2 to 8 hours) and concomitant with maximal uveitis (18 to 24 hours). Low levels of TNF and IL-6 were found in aqueous humor of BN rats. Ocular IL-6 mRNA was detected at the same time as IL-6 activity was measured in aqueous humor.

CONCLUSIONS

The results of this study indicate that both TNF and IL-6 may play a role in the pathogenesis of EIU. The early release of TNF in aqueous humor during EIU suggests that this cytokine may serve as an initial mediator of intraocular inflammation. Furthermore, Northern blot analysis indicates that IL-6 is produced locally during EIU.

<em>IL</em>-8 plays an integral role in promoting the malignant phenotype in breast cancer, and its production is directly influenced by inflammatory cytokines in the tumor microenvironment. Here, we show that activation of <em>IL</em>-1beta receptors on malignant HS578t and MDA-MB-231 breast cancer cells strongly induces <em>IL</em>-8 expression and that RNA stabilization is persistently activated at least 12-24 hr after stimulation. SB <em>20</em>3580 and rapamycin reversed the RNA stabilization effect of <em>IL</em>-1beta in a dose-dependent manner, suggesting involvement of the p38/MAP kinase and mTOR pathways. A luciferase reporter assay indicated that the stabilization effect was dependent on cis elements in the 3'-untranslated region (UTR) of the <em>IL</em>-8 transcript. By UV cross-linking, we identified multiple cellular factors that interact with the <em>IL</em>-8 3'UTR, ranging 34-76 kDa. Immunoprecipitation analysis indicated that HuR, KSRP and TIAR bound to one or more loci in the 3'UTR. While the cross-linking patterns were similar, quantitative immunoprecipitation of native <em>IL</em>-8 RNA from <em>IL</em>-1beta-stimulated cytoplasmic extract revealed a <em>20</em>-fold greater association of transcript with the stabilizing factor HuR vs. the destabilizing factor KSRP. In conclusion, <em>IL</em>-1beta is a potent cytokine stimulus for <em>IL</em>-8 RNA stabilization in breast cancer cells, possibly by enhanced binding of cytoplasmic HuR to the 3'UTR.

Newly discovered <em>IL</em>-9-producing helper T cells (Th9) reportedly exert both aggravating and suppressive roles on experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, it is still unclear whether Th9 is a distinct Th cell subset and how <em>IL</em>-9 functions in the CNS. In this study, we show that <em>IL</em>-9 is produced by naive CD4(+) T cells that were stimulated with anti-CD3 and anti-CD28 Abs under the conditions of Th2-, inducible regulatory T cell-, Th17-, and Th9-polarizing conditions and that <em>IL</em>-9 production is significantly suppressed in the absence of <em>IL</em>-4, suggesting that <em>IL</em>-4 is critical for the induction of <em>IL</em>-9 by each producing cell. The <em>IL</em>-9 receptor complex, <em>IL</em>-9R and <em>IL</em>-2Rγ, is constitutively expressed on astrocytes. <em>IL</em>-9 induces astrocytes to produce CCL-<em>20</em> but not other chemokines, including CCL-2, CCL-3, and CXCL-2 by astrocytes. The conditioned medium of <em>IL</em>-9-stimulated astrocytes induces Th17 cell migration in vitro, which is cancelled by adding anti-CCL-<em>20</em> neutralizing Abs. Treating with anti-<em>IL</em>-9 neutralizing Abs attenuates experimental autoimmune encephalomyelitis, decreases the number of infiltrating Th17 cells, and reduces CCL-<em>20</em> expression in astrocytes. These results suggest that <em>IL</em>-9 is produced by several Th cell subsets in the presence of <em>IL</em>-4 and induces CCL-<em>20</em> production by astrocytes to induce the migration of Th17 cells into the CNS.

OBJECTIVE

This study investigated the effects of a dietary supplement on exercise-induced markers of cell damage and the inflammatory mediators C-reactive protein (CRP) and interleukin-6 (IL-6).

METHODS

The supplement contained mixed tocopherols, flavonoids, and docosahexaenoate. Forty healthy, nonsmoking, untrained males (aged 18-35 yr) were randomly assigned to receive either the supplement (N = 20) or placebo (N = 20) during the 14-d experimental protocol. Blood samples were collected on day 0 (baseline), day 7 (eccentric exercise-induced injury), day 10, and day 14.

OBJECTIVE

Markers of cell damage (creatine kinase (CK) and lactate dehydrogenase (LDH)) and inflammation IL-6 and CRP were assessed at these time points in conjunction with subjective range of motion (ROM) and perceived pain measurements. Statistical analyses were conducted using nonparametric methods (P < 0.05).

RESULTS

Eccentric arm curl exercise was used to induce an acute phase injury response as evidenced by significant (P < 0.0001) increases in CK, LDH, and pain, as well as a decreased range of motion 3 d after the exercise. There were no significant differences between groups in CK and LDH responses. In contrast, there were significant group differences for IL-6 (P = 0.008) and CRP (P = 0.003). At day 10, by Mann-Whitney U test of changes, the placebo group had significantly greater increases in IL-6 and CRP than the treatment group (P = 0.05 and P < 0.01), respectively.

CONCLUSIONS

This study suggested that exercise-induced inflammation, evaluated by changes in IL-6 and CRP, was significantly reduced by the dietary supplement.

Interleukin-15 (<em>IL</em>-15) is produced by human bone marrow (BM) stromal cells and can induce CD34(+) hematopoietic progenitor cells (HPCs) to differentiate into CD56(+)CD3(-) natural killer (NK) cells in the absence of stromal cells. <em>IL</em>-15 mediates its effects by signaling through the beta and gammac chains of the <em>IL</em>-2/15 receptor (R). The c-kit ligand (KL), also produced by stromal cells, enhances the expansion of NK cells from CD34(+) HPCs in the presence of <em>IL</em>-15, but alone has no ability to differentiate NK cells. Mice deficient in KL do not appear to have a quantitative deficiency in NK cells, suggesting that other stromal cell factors may contribute to NK cell expansion. Flt3 ligand (FL) is also produced by BM stromal cells and has homology with KL. Furthermore, mice with a targeted disruption of the FL gene have reduced numbers of NK cells. We evaluated here the effects of FL on human NK cell development and expansion from CD34(+) HPCs. Like KL, FL significantly enhanced the expansion of NK cells from CD34(+) HPCs in the presence of <em>IL</em>-15, compared with <em>IL</em>-15 alone. However, FL alone had no effect on NK cell differentiation. We therefore explored the mechanism by which FL promotes <em>IL</em>-15-mediated NK cell development. FL was found to induce <em>IL</em>-2/15Rbeta (CD122) expression on CD34(bright) HPCs. The CD34(bright) CD122(+) cell coexpressed CD38, but lacked expression of CD7, CD56, NK cell receptors (NKRs), or cytotoxic activity in the absence of <em>IL</em>-15. Using limiting dilution analysis in the presence of <em>IL</em>-15 alone, we demonstrated that the FL-induced CD34(bright)CD122(+) HPCs had an NK cell precursor frequency <em>20</em>- to 60-fold higher than the CD34(dim/neg)CD122(-) HPCs and 65- to 235-fold higher than fresh CD34(+) HPCs. KL had similar effects as FL, but induced a significantly lower percentage of CD34(bright)CD122(+) cells (P </=.01). Both FL and KL also increased <em>IL</em>-15R transcript in CD34(+) HPCs. Culture of CD34(+) HPCs in FL or KL, followed by culture in <em>IL</em>-15 alone, induced expression of both C-type lectin and Ig-superfamily NKRs on CD56(+) cells. These data collectively support a role for FL in early human NK cell development. FL or KL generate a unique CD34(bright) CD122(+)CD38(+) human NK cell intermediate from CD34(+) HPCs that lacks NK features yet is <em>IL</em>-15-responsive. <em>IL</em>-15 is then required for the induction of CD56 and NKRs, LGL morphology, cytotoxic activity, and the ability to produce abundant cytokines and chemokines.

BACKGROUND

Receptor antagonists that block the binding of chemokines such as CXCL8 (IL-8) are effective in animals models of neutrophil-mediated inflammation. It has been hypothesized that selective inhibition of neutrophil trafficking and activation may be a useful adjunct for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease or cystic fibrosis. A CXCR1/2 receptor antagonist has shown activity in an ozone challenge model in humans.

CONCLUSIONS

SB-656933, a selective CXCR2 antagonist, is safe and well-tolerated at single doses and is shown to inhibit agonist (CXCL1)-mediated expression of the CD11b on peripheral blood neutrophils as well as ozone-induced airway neutrophilia in healthy subjects.

OBJECTIVE

To determine the safety and tolerability of a novel selective CXCR2 antagonist and assess its pharmacodynamic effects using measures of neutrophil activation and function, including CD11b expression in whole blood and ozone-induced airway inflammation in healthy subjects.

METHODS

Flow cytometric determination of ex vivo CXCL1-induced CD11b expression on peripheral blood neutrophils was performed following single dose oral administration of SB-656933 (dose range 2-1100 mg). A subsequent randomized study (placebo, 50 mg and 150 mg) was performed to explore the dose-response for ozone-induced airway inflammation, as measured by sputum biomarkers.

RESULTS

Oral administration of SB-656933 resulted in significant inhibition of CXCL1-induced CD11b expression on peripheral blood neutrophils at single doses greater than or equal to 50 mg. Maximum inhibition (70%) relative to placebo was observed following administration of SB-656933 400 mg (95% CI 60%, 77%). This was sustained up to a dose of 1100 mg. Single doses of SB-656933 reduced ozone-induced airway inflammation in a dose-dependent manner. Relative to placebo, there were 55% (95% CI 20%, 75%) and 74% (95% CI 55%, 85%) fewer neutrophils in the sputum of subjects after a single dose of 50 mg or 150 mg, respectively. There was a corresponding reduction in myeloperoxidase concentrations in the sputum supernatant of 32.8% (95% CI 9.2, 50.3) and 50.5% (95% CI 33.3, 63.3). SB-656933 was safe and well-tolerated at all doses.

CONCLUSIONS

SB-656933 is a CXCR2 antagonist that demonstrates dose-dependent effects on neutrophil activation and recruitment within a well-tolerated dose range. These data suggest that SB-656933 may be an effective agent in neutrophil-predominant diseases.

The expression levels of mRNA encoding a panel of 28 chicken cytokines and chemokines were quantified in intestinal lymphocytes following Eimeria acervulina and Eimeria tenella primary and secondary infections. Compared with uninfected controls, transcripts of the pro-inflammatory cytokines IFN-alpha, <em>IL</em>-1beta, <em>IL</em>-6, and <em>IL</em>-17 were increased up to <em>20</em><em>20</em>-fold following primary infection. By contrast, following secondary infection by either microorganism, pro-inflammatory mRNAs levels were relatively unchanged (< or = <em>20</em>-fold). Transcripts encoding the Th1 and Th1 regulatory cytokines IFN-gamma, <em>IL</em>-2, <em>IL</em>-10, <em>IL</em>-12, <em>IL</em>-15, <em>IL</em>-16, and <em>IL</em>-18 were uniformly increased 14-2471-fold after E. acervulina primary infection, but either unchanged (<em>IL</em>-15, <em>IL</em>-16, <em>IL</em>-18), increased (IFN-gamma, <em>IL</em>-10, <em>IL</em>-12), or decreased (<em>IL</em>-2) following E. tenella primary infection. Following secondary infections, Th1 cytokine mRNA levels were relatively unchanged, with the exception of <em>IL</em>-12 which was increased 1.5 x 10(5)-fold after E. acervulina and decreased 5.1 x 10(4)-fold after E. tenella infection. Transcripts for the Th2 or Th2 regulatory cytokines <em>IL</em>-3 and GM-CSF were increased up to 327-fold following primary or secondary infection with both parasites, while <em>IL</em>-4 and <em>IL</em>-13 mRNAs were decreased 25- to 2 x 10(5)-fold after primary or secondary infection. The dynamics of chicken chemokine expression revealed modest changes (<100-fold) following primary or secondary infection except for lymphotactin. When lymphocyte subpopulations were similarly analyzed, IFN-gamma, <em>IL</em>-2, <em>IL</em>-3, <em>IL</em>-15, and MIF were most highly increased in TCR2(+) cells following E. acervulina infection, while TCR1(+) cells only expressed high levels of <em>IL</em>-16 following E. tenella infection. In contrast, CD4(+) cells only expressed highest levels of <em>IL</em>-10 after E. acervulina infection, whereas these cells produced abundant transcripts for IFN-gamma, <em>IL</em>-3, <em>IL</em>-15, and MIF after E. tenella infection. We conclude that coccidiosis induces a diverse and robust primary cytokine/chemokine response, but a more subdued secondary response.

The signal transduction pathways regulating smooth-muscle gene expression and production of cytokines in response to proinflammatory mediators are undefined. Cultured human bronchial smooth-muscle cells were treated for <em>20</em> h with a cytokine cocktail containing interleukin (<em>IL</em>)-1beta, tumor necrosis factor-alpha, and interferon-gamma. A complementary DNA expression array containing 588 genes was used to follow cytokine-stimulated gene expression. The expression and secretion of the cytokines <em>IL</em>-1beta, <em>IL</em>-6, and <em>IL</em>-8 significantly increased after <em>20</em> h of stimulation as measured by relative reverse transcriptase/ polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting techniques. Expression of <em>IL</em>-6 and <em>IL</em>-8 was sensitive to SB<em>20</em>3580, the specific inhibitor of p38 mitogen-activated protein (MAP) kinase and PD98059, an inhibitor of MAP kinase kinase. Expression of <em>IL</em>-1beta was sensitive only to PD98059. Together, these results demonstrate that the p38 and extracellular signal-regulated protein kinase MAP kinase pathways are required for proinflammatory mediator- induced cytokine expression in airway myocytes. The generation of chemokines and cytokines in airway smooth muscle also provides evidence that smooth-muscle cells have the ability to contribute to the inflammatory response.

OBJECTIVE

Many patients with septic shock and increased cardiac troponin I (cTnI) do not exhibit significant left ventricular systolic dysfunction. We hypothesized that an isolated and reversible impairment of ventricular relaxation may be associated with the increase in cTnI.

METHODS

Prospective, observational study.

METHODS

Surgical intensive care unit in a university hospital.

METHODS

Total of 54 patients with septic shock.

METHODS

Fractional area change, early diastolic velocity of mitral annulus, flow propagation velocity of early diastolic mitral inflow, cTnI, tumor necrosis factor-alpha, interleukin (IL)-6, -1beta, -8, and -10 were measured at days 1, 2, 3, 4, 7, and 10 after onset of septic shock. Patients were classified into three groups: normal cTnI (group 1), increased cTnI and fractional area change <50% (group 2), and increased cTnI and fractional area change >50% (group 3).

RESULTS

A total of 22 patients had an increase in cTnI, 11 with both systolic and diastolic dysfunctions and 11 with isolated impairment of left ventricular relaxation. At day 1, early diastolic velocity of mitral annulus and flow propagation velocity of early diastolic mitral inflow were significantly lower and tumor necrosis factor-alpha, IL-8, and IL-10 significantly higher in groups 2 and 3 compared with group 1. With resolution of septic shock, early diastolic velocity of mitral annulus and flow propagation velocity of early diastolic mitral inflow measured in patients of groups 2 and 3 returned progressively to values observed in group 1, with a parallel normalization of tumor necrosis factor-alpha, IL-8, and IL-10.

CONCLUSIONS

Isolated and reversible impairment of left ventricular relaxation, associated with transient increases in cTnI, tumor necrosis factor-alpha, IL-8, and IL-10, was observed in 20% of patients with septic shock.

<em>IL</em>-18 expression has recently been detected in rheumatoid arthritis (RA) synovial membrane. We investigated the mechanisms by which <em>IL</em>-18-induced collagen-induced arthritis in DBA/1 mice primed intradermally with type II bovine collagen in IFA and boosted i.p. 21 days later with CII in saline. Mice were injected i.p. with r<em>IL</em>-12, r<em>IL</em>-18, or both (100 ng) during days -1 to 4 and again on days <em>20</em>-24. Control mice received PBS. Mice treated with <em>IL</em>-12 or <em>IL</em>-18 alone developed significantly higher incidence and more severe disease compared with controls. These were elevated further by combination treatment with <em>IL</em>-12 and <em>IL</em>-18. The cytokine treatments led to markedly enhanced synovial hyperplasia, cellular infiltration, and cartilage erosion compared with controls. Cytokine-treated mice produced significantly more IFN-gamma, TNF-alpha, and <em>IL</em>-6 than the controls. Interestingly, <em>IL</em>-18-treated mice produced more TNF-alpha and <em>IL</em>-6, but less IFN-gamma, compared with mice treated with <em>IL</em>-12. Furthermore, splenic macrophages from DBA/1 mice cultured in vitro with <em>IL</em>-18, but not <em>IL</em>-12, produced substantial amounts of TNF-alpha. Mice treated with <em>IL</em>-18 or <em>IL</em>-18 plus <em>IL</em>-12 produced markedly more IgG1 and IgG2a anti-collagen Ab compared with controls, whereas <em>IL</em>-12 treatment only led to an enhanced IgG2a response. Together these results demonstrate that <em>IL</em>-18 can promote collagen-induced inflammatory arthritis through mechanisms that may be distinct from those induced by <em>IL</em>-12.