The present study investigated bystander suppression, specific suppression and anergy as mechanisms for oral tolerance. Oral tolerance was induced in mice by a single gastric intubation of <em>20</em> mg ovalbumin (OVA) and was evaluated in vitro by the absence of T lymphocyte proliferative responses to OVA after priming by OVA-complete Freund's adjuvant (CFA). T lymphocyte unresponsiveness was antigen specific, systemic and was not affected by the vehicle used for immunization. T lymphocytes derived from tolerant popliteal lymph nodes (PLN) responded to an acetone precipitate (AP) of mycobacteria present in CFA; this response was not suppressed by co-culture with OVA, thereby arguing against a mechanism of bystander suppression in our system. Responses of PLN T lymphocytes derived from OVA-CFA primed, non-tolerant mice, or those of an OVA-specific T lymphocyte line, were not suppressed by PLN or spleen cells derived from OVA tolerant mice. These results excluded the possibility that oral tolerance was induced and maintained by a mechanism of specific suppression. At the cellular level, we found that OVA-tolerant T lymphocytes did not produce interleukin-2 (<em>IL</em>-2) nor express <em>IL</em>-2 receptor in response to OVA stimulation in vitro; both observations are indicative of a state of anergy. Incubation of OVA-tolerant PLN T lymphocytes together with murine recombinant <em>IL</em>-2 for 5 days, released anergic T lymphocytes and a concomitant OVA-specific proliferative response of CD4+ T cells was detected. Taken together, our experimental system excludes the involvement of bystander or specific suppression in the induction of oral tolerance to OVA, and provides direct evidence to show that oral tolerance results from specific T lymphocyte anergy.

BACKGROUND

Chronic rhinosinusitis (CRS) is one of the most common chronic diseases. Its etiology is unknown, and there is a paucity of effective medical treatments.

OBJECTIVE

We tested the hypothesis that intranasal antifungal treatment improves the objective computed tomography (CT) findings (inflammatory mucosal thickening), nasal endoscopy stages, and symptoms of CRS.

METHODS

A randomized, placebo-controlled, double-blind, single-center trial used amphotericin B to treat 30 randomly selected patients with CRS. Patients were instructed to instill <em>20</em> mL amphotericin B (250 mug/mL) or placebo to each nostril twice daily for 6 months. The primary outcome was a quantitative reduction in inflammatory mucosal thickening on CT scans of a standardized coronal cut. Secondary outcome measures were endoscopic scores, patient symptom scores, and levels of intranasal inflammatory mediators.

RESULTS

Twenty-four patients completed the 6 months of treatment. Patients receiving amphotericin B achieved a relative reduction in the percentage of mucosal thickening on CT scans (n = 10; -8.8%) compared with placebo (n = 14; +2.5%; P = .030). Likewise, the changes in the endoscopic scores improved in the amphotericin B group compared with placebo ( P = .038). Between-group comparisons of the changes in the intranasal mucus levels of eosinophil-derived neurotoxin showed a reduction in the amphotericin B group and an increase in the placebo group ( P = .046); levels of IL-5 showed similar tendencies ( P = .082).

CONCLUSIONS

Intranasal amphotericin B reduced inflammatory mucosal thickening on both CT scan and nasal endoscopy and decreased the levels of intranasal markers for eosinophilic inflammation in patients with CRS.

Hepatitis C virus (HCV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), a life-threatening sequel. However, the factors that affect disease progression to HCC have not been thoroughly elucidated. Genetic polymorphisms in proinflammatory cytokines, the interleukin 1 (<em>IL</em>-1) family (<em>IL</em>-1beta and <em>IL</em>-1ra) and tumor necrosis factor-alpha (TNF-alpha), were studied in 274 Japanese patients with chronic HCV infection and 55 healthy individuals using standard polymerase chain reaction-based genotyping techniques. The association between these polymorphisms and disease status was evaluated while controlling for confounding clinical variables. The proportion of patients with HCC in the <em>IL</em>-1beta-31 T/T (55%, odds ratio to C/C was 2.63, P =.009) genotype was higher than in the T/C (44%, odds ratio to C/C was 1.64, P =.149) and C/C genotypes (35%). The <em>IL</em>-1beta-31 and -511 loci were in near complete linkage disequilibrium, and the <em>IL</em>-1beta-511/-31 haplotype C-T was significantly associated with the presence of HCC (odds ratio of 1.51, P =.02). Polymorphisms in the TNF-alpha gene were not associated with disease. A multivariate analysis revealed that the <em>IL</em>-1beta-31 T/T genotype, alpha-fetoprotein>><em>20</em> microg/L, presence of cirrhosis, male sex, and age >60 years were associated with the presence of HCC at odds ratios of 3.73 (T/T vs. C/C), 4.12, 4.03, 3.89, and 3.27, respectively. In conclusion, the <em>IL</em>-1beta-31 genotype T/T or the <em>IL</em>-1beta-511/-31 haplotype C-T is associated with the presence of HCC in Japanese patients with chronic HCV infection.

By using a sandwich ELISA, soluble human <em>IL</em>-6 receptor (s<em>IL</em>-6 R) levels were measured in the sera of <em>20</em> healthy children and of 25 patients with systemic juvenile rheumatoid arthritis (JRA). In patients with systemic JRA, serum s<em>IL</em>-6 R levels (114.6 +/- 37.7 ng/ml) were significantly lower (P < 0.01) than those of healthy children (161.2 +/- 45.5 ng/ml). Serum s<em>IL</em>-6 R levels were negatively correlated (r = -0.610, P < 0.001) with serum <em>IL</em>-6 levels measured with the B9 cells. The serum <em>IL</em>-6/s<em>IL</em>-6 R complex was detected using an ELISA based on a monoclonal antibody to <em>IL</em>-6 for capture and on a monoclonal antibody to human s<em>IL</em>-6 R for detection. Healthy controls had little, if any, detectable serum <em>IL</em>-6/s<em>IL</em>-6 R complex (OD 0.024 +/- 0.027), while the majority of patients with systemic JRA presented measurable serum <em>IL</em>-6/s<em>IL</em>-6 R complex (OD 0.492 +/- 0.546). <em>IL</em>-6 levels estimated in the circulating <em>IL</em>-6/s<em>IL</em>-6 R complexes were in the range of nanograms per milliliter and approximately <em>20</em>-fold higher than those measured by the B9 cells. Since serum C-reactive protein concentrations were much more correlated with serum levels of <em>IL</em>-6/s<em>IL</em>-6 R complexes (r = 0.713, r2 = 0.51, P < 0.0001) than with the serum <em>IL</em>-6 levels measured with the B9 cells (r = 0.435, r2 = 0.19, P = 0.05), the large quantities of serum <em>IL</em>-6 present in <em>IL</em>-6/s<em>IL</em>-6 R complexes appear to be biologically relevant in vivo, at least as far as the induction by <em>IL</em>-6 of acute phase protein production.

The objective of the present study was to investigate the effect of leptin, alone or in combination with <em>IL</em>-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and <em>IL</em>-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and <em>IL</em>-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 micromol/l] and LY294002 [1, 2.5, 5 and 10 micromol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, <em>20</em> and 30 micromol/l]) and p38 kinase (SB<em>20</em>3580 [1, 5, 10, <em>20</em> and 30 micromol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with <em>IL</em>-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and <em>IL</em>-1 resulted in a net increase in nitric oxide concentration over <em>IL</em>-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/<em>IL</em>-1. Finally, wortmannin, LY294002, PD098059 and SB<em>20</em>3580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5 chondrocytes, and in human primary chondrocytes. This study indicates that leptin plays a proinflammatory role, in synergy with <em>IL</em>-1, by inducing NOS type II through a signalling pathway that involves JAK2, PI3K, MEK-1 and p38 kinase.

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2'-5' oligoadenylate synthetases. 2'-5' oligoadenylate synthetase is stimulated by dsRNA to produce 5'-phosphorylated, 2'-5'-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription >>/=<em>20</em>-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 muM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, <em>IL</em>-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-beta superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.

The poor prognosis for patients with advanced malignancy relates partly to the inability to reverse cancer metastasis. In this study we have investigated an integrated immunotherapy method against pre-established metastases in three kinds of advanced cancer models including B16 melanoma, 4T1 breast tumor, and Hca hepatoma. The progression of metastases into multistep lymph nodes (LN) and internal organs was, markedly impeded in the midway stage and reversed in the ultimate stage following a <em>20</em>-day course of intravenous immunotherapy [with interleukin-12 (<em>IL</em>-12) gene-engineered mesenchymal stem cells (MSCs), administered once every 5 days P < 0.05)]; the therapy was without systemic toxic effects. As the control, obvious systemic toxicity was observed in the free Ad<em>IL</em>-12 group, yet metastasis was partly delayed only in the midway stage but not in the ultimate stage. Enzyme-linked immunosorbent assay (ELISA) showed that the intratumoral expression levels of <em>IL</em>-12 were enhanced by cytokine-engineered MSCs to be tenfold greater than that of free Ad<em>IL</em>-12 groups in the ultimate stage; conversely, free Ad<em>IL</em>-12 groups showed elevated serum, but not intratumoral levels of <em>IL</em>-12, during the midway stage. Furthermore, histomorphometric analysis revealed a reductive tendency toward reversion of tumor-associated lymphatic sprouts and an increased tumor apoptosis index in engineered MSC groups (P < 0.05). These data indicate the potential of cytokine-engineered MSCs to be considered as an integrated therapeutic weapon for targeting advanced malignancies.

Numerous studies have reported altered levels of in vitro production of the cytokines interleukin-1 (<em>IL</em>-1) and tumor necrosis factor (TNF) from blood leukocytes in various human disease states. Most of these studies have used bioassays which are vulnerable to inhibitors produced by these cells. Furthermore in vitro cytokine production is often assessed on a single occasion. The present study was designed to standardize stimulation conditions for in vitro <em>IL</em>-1 beta production and to employ a competitive radioimmunoassay (RIA) to demonstrate reproducibility and long-term variation of in vitro cytokine production in a cohort of healthy human subjects. We also examined relative amounts of immunoreactive <em>IL</em>-1 beta, <em>IL</em>-1 alpha, and TNF induced by the stimuli endotoxin, phytohemagglutinin, or Staphylococcus epidermidis. We show that the RIA can reliably detect <em>IL</em>-1 beta produced from mononuclear cells in concentrations as low as 115 pg/ml. Lysing cells by repeated freeze-thawing yields maximal recovery of total (i.e., secreted plus cell-associated) immunoreactive <em>IL</em>-1 beta, when compared to extraction with the detergent CHAPS or addition of protease inhibitors. Repeated measurement of in vitro cytokine production on different days within 1 week shows good reproducibility for a given individual and a given stimulus (variation coefficient <em>20</em> to 30%). Over a long time period (6 months) in vitro cytokine production is stable in some individuals but changes considerably in others. The soluble stimulus endotoxin induces twofold more <em>IL</em>-1 alpha than <em>IL</em>-1 beta or TNF; in contrast the phagocytic stimulus heat-killed S. epidermidis induces fourfold more <em>IL</em>-1 beta and TNF than <em>IL</em>-1 alpha. This distinct pattern of cytokine response indicates differential stimulation of the mononuclear cells by different stimuli. The results form the basis for studying in vitro cytokine production in different human disease states.

The essentiality of n-6 polyunsaturated fatty acids (PUFA) is described in relation to a thymus/thymocyte accretion of arachidonic acid (<em>20</em>:4n-6, AA) in early development, and the high requirement of lymphoid and other cells of the immune system for AA and linoleic acid (1 8:2n-6, LA) for membrane phospholipids. Low n-6 PUFA intakes enhance whereas high intakes decrease certain immune functions. Evidence from in vitro and in vivo studies for a role of AA metabolites in immune cell development and functions shows that they can limit or regulate cellular immune reactions and can induce deviation toward a T helper (Th)2-like immune response. In contrast to the effects of the oxidative metabolites of AA, the longer-chain n-6 PUFA produced by gamma-linolenic acid (18:3n-6, GLA) feeding decreases the Th2 cytokine and immunoglobulin (Ig)G1 antibody response. The n-6 PUFA, GLA, dihomo-gamma-linolenic acid (<em>20</em>:3n-6, DHLA) and AA, and certain oxidative metabolites of AA can also induce T-regulatory cell activity, e.g., transforming growth factor (TGF)-beta-producing T cells; GLA feeding studies also demonstrate reduced proinflammatory interleukin (<em>IL</em>)-1 and tumor necrosis factor (TNF)-alpha production. Low intakes of long-chain n-3 fatty acids (fish oils) enhance certain immune functions, whereas high intakes are inhibitory on a wide range of functions, e.g., antigen presentation, adhesion molecule expression, Th1 and Th2 responses, proinflammatory cytokine and eicosanoid production, and they induce lymphocyte apoptosis. Vitamin E has a demonstrable critical role in long-chain n-3 PUFA interactions with immune functions, often reversing the effects of fish oil. The effect of dietary fatty acids on animal autoimmune disease models depends on both the autoimmune model and the amount and type of fatty acids fed. Diets low in fat, essential fatty acid deficient (EFAD), or high in long-chain n-3 PUFA from fish oils increase survival and reduce disease severity in spontaneous autoantibody-mediated disease, whereas high-fat LA-rich diets increase disease severity. In experimentally induced T cell-mediated autoimmune disease, EFAD diets or diets supplemented with long-chain n-3 PUFA augment disease, whereas n-6 PUFA prevent or reduce the severity. In contrast, in both T cell- and antibody-mediated autoimmune disease, the desaturated/elongated metabolites of LA are protective. PUFA of both the n-6 and n-3 families are clinically useful in human autoimmune-inflammatory disorders, but the precise mechanisms by which these fatty acids exert their clinical effects are not well understood. Finally, the view that all n-6 PUFA are proinflammatory requires revision, in part, and their essential regulatory and developmental role in the immune system warrants appreciation.

OBJECTIVE

The antiinflammatory effect of macrolide antibiotics has been well-established, as has their role in the treatment of certain disorders of chronic airway inflammation. Several studies have suggested that long-term, low-dose macrolides may be efficacious in the treatment of chronic rhinosinusitis; however, these studies have lacked a control group. To date, this effect has not been tested in a randomized, placebo-controlled study.

METHODS

The authors conducted a double-blind, randomized, placebo-controlled clinical trial on 64 patients with chronic rhinosinusitis. Subjects received either 150 mg roxithromycin daily for 3 months or placebo. Outcome measures included the Sinonasal Outcome Test-<em>20</em> (SNOT-<em>20</em>), measurements of peak nasal inspiratory flow, saccharine transit time, olfactory function, nasal endoscopic scoring, and nasal lavage assays for interleukin-8, fucose, and a2-macroglobulin.

RESULTS

There were statistically significant improvements in SNOT-<em>20</em> score, nasal endoscopy, saccharine transit time, and IL-8 levels in lavage fluid (P<.05) in the macrolide group. A correlation was noted between improved outcome measures and low IgE levels. No significant improvements were noted for olfactory function, peak nasal inspiratory flow, or lavage levels for fucose and a2-macroglobulin. No improvement in any outcome was noted in the placebo-treated patients.

CONCLUSIONS

These findings suggest that macrolides may have a beneficial role in the treatment of chronic rhinosinusitis, particularly in patients with low levels of IgE, and supports the in vitro evidence of their antiinflammatory activity. Additional studies are required to assess their place in clinical practice.

BACKGROUND

Low-T(3) syndrome is a predictor of poor outcome in patients with cardiac dysfunction. The study aimed to assess the short-term effects of synthetic L-T(3) replacement therapy in patients with low-T(3) syndrome and ischemic or nonischemic dilated cardiomyopathy (DC).

METHODS

A total of <em>20</em> clinically stable patients with ischemic (n = 12) or nonischemic (n = 8) DC were enrolled. There were 10 patients (average age 72 yr, range 66-77; median, 25-75th percentile) who underwent 3-d synthetic L-T(3) infusion (study group); the other 10 patients (average age 68 yr, range 64-71) underwent placebo infusion (control group). Clinical examination, electrocardiography, cardiac magnetic resonance, and bio-humoral profile (free thyroid hormones, TSH, plasma renin activity, aldosterone, noradrenaline, N-terminal-pro-B-Type natriuretic peptide, and <em>IL</em>-6) were assessed at baseline and after 3-d synthetic L-T(3) (initial dose: <em>20</em> microg/m(2) body surface.d) or placebo infusion.

RESULTS

After T(3) administration, free T(3) concentrations increased until reaching a plateau at 24-48 h (3.43, 3.<em>20</em>-3.84 vs. 1.74, 1.62-1.93 pg/ml; P = 0.03) without side effects. Heart rate decreased significantly after T(3) infusion (63, 60-66 vs. 69, 60-76 beats per minute; P = 0.008). Plasma noradrenaline (347; 270-740 vs. 717, 413-808 pg/ml; P = 0.009), N-terminal pro-B-Type natriuretic peptide (3000, 438-4005 vs. 3940, 528-5628 pg/ml; P = 0.02), and aldosterone (175, 152-229 vs. 231, 154-324 pg/ml; P = 0.047) significantly decreased after T(3) administration. Neurohormonal profile did not change after placebo infusion in the control group. After synthetic L-T(3) administration, left-ventricular end-diastolic volume (142, 132-161 vs. 133, 114-158 ml/m(2) body surface; P = 0.02) and stroke volume (40, 34-44 vs. 35, 28-39 ml/m(2) body surface; P = 0.01) increased, whereas external and intracardiac workload did not change.

CONCLUSIONS

In DC patients, short-term synthetic L-T(3) replacement therapy significantly improved neuroendocrine profile and ventricular performance. These data encourage further controlled trials with more patients and longer periods of synthetic L-T(3) administration.

Pro- and antiinflammatory cytokines and mediators were measured in 39 patients with acute life-threatening meningococcal infections classified into 3 groups: A, meningitis without shock (n = <em>20</em>); B, meningitis with shock (n = 9); and C, shock without meningitis (n = 10). The plasma concentrations of proinflammatory endotoxin, tumor necrosis factor-alpha (TNF-alpha), interleukin (<em>IL</em>)-6, and <em>IL</em>-8 and antiinflammatory cytokines and mediators <em>IL</em>-1 receptor antagonist, <em>IL</em>-10, and soluble TNF receptors p55 and p75 were strongly associated with this classification; the highest concentrations were in group C. <em>IL</em>-4 was not measurable. <em>IL</em>-1 beta was increased only in rapidly fatal cases. In addition, cerebrospinal fluid (CSF) was analyzed in 21 patients for TNF-alpha and its soluble receptors. In CSF, these compounds were mainly increased in group A, reflecting an intrathecal compartmentalized cytokine production. It is concluded that both pro- and antiinflammatory mediators are simultaneously increased and are strongly associated with a classification based on simple clinical parameters.

Tricyclic antidepressants (TCAs) are widely used in treating depressive disorders. It has been demonstrated that, for instance, <em>IL</em>-1 beta and <em>IL</em>-6 inhibit the HPA axis, which plays a role in the development of depressions. Therefore. we were interested in investigating how TCAs influence cytokine release by T lymphocytes and monocytes respectively. Cells were incubated with either 5 microM clomipramine, 15 microM imipramine or <em>20</em> microM citalopram. <em>IL</em>-2 release was suppressed to 60% of the control values by clomipramine and imipramine (p = 0.001; p = 0.000), but citalopram was found to cause a much weaker inhibition (only 18%) (p = 0.16). INF-gamma release was affected to a lower degree than <em>IL</em>-2 release, and imipramine (34%) (p = 0.054) was more potent than clomipramine (24%) (p = 0.16) and citalopram (12%) (p = 0.059) in this case. Monocytes incubated with TCA for 4 h exhibited only limited inhibition of <em>IL</em>-1 beta and <em>IL</em>-6 release, i.e., 6-25% for all three compounds. The corresponding value for TNF-alpha release was <em>20</em>-45% inhibition, with citalopram being the weakest inhibitor. After 10 h of monocytes to LPS exposure, all three compounds exerted a strong inhibition of <em>IL</em>-1 beta and TNF-alpha release, i.e., 60-70% with p-values below 0.012 for all of them. However the inhibition of <em>IL</em>-6 release was less than 35%. Citalopram was equality as potent as imipramine and clomipramine in inhibiting <em>IL</em>-6 release after long-term exposure of monocytes to LPS. All three TCAs elevated intracellular cAMP concentrations significantly in T lymphocytes and monocytes (p < 0.001).

BACKGROUND

Observational studies link statin therapy with improved outcomes in patients with severe sepsis.

OBJECTIVE

To test whether atorvastatin therapy affects biologic and clinical outcomes in critically ill patients with severe sepsis.

METHODS

Phase II, multicenter, prospective, randomized, double-blind, placebo-controlled trial stratified by site and prior statin use. A cohort of 250 critically ill patients (123 statins, 127 placebo) with severe sepsis were administrated either atorvastatin (<em>20</em> mg daily) or matched placebo.

RESULTS

There was no difference in IL-6 concentrations (primary end point) between the atorvastatin and placebo groups (P = 0.76) and no interaction between treatment group and time to suggest that the groups behaved differently over time (P = 0.26). Baseline plasma IL-6 was lower among previous statin users (129 [87-191] vs. 244 [187-317] pg/ml; P = 0.01). There was no difference in length of stay, change in Sequential Organ Failure Assessment scores or mortality at intensive care unit discharge, hospital discharge, 28- or 90-day (15% vs. 19%), or adverse effects between the two groups. Cholesterol was lower in patients treated with atorvastatin (2.4 [0.07] vs. 2.6 [0.06] mmol/L; P = 0.006). In the predefined group of 77 prior statin users, those randomized to placebo had a greater 28-day mortality (28% vs. 5%; P = 0.01) compared with those who received atorvastatin. The difference was not statistically significant at 90 days (28% vs. 11%; P = 0.06).

CONCLUSIONS

Atorvastatin therapy in severe sepsis did not affect IL-6 levels. Prior statin use was associated with a lower baseline IL-6 concentration and continuation of atorvastatin in this cohort was associated with improved survival. Clinical trial registered with the Australian New Zealand Clinical Trials Registry (ACTRN 12607000028404).

OBJECTIVE

We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models.

METHODS

The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model.

RESULTS

Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (<em>20</em>-3,500 nmol/L). Ganetespib was also approximately <em>20</em>-fold more potent in isogenic Ba/F3 pro-B cells rendered <em>IL</em>-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA.

CONCLUSIONS

Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.

Carbon nanotubes (CNTs) are novel materials with unique electronic and mechanical properties. The extremely small size, fiberlike shape, large surface area, and unique surface chemistry render their distinctive chemical and physical characteristics and raise potential hazards to humans. Several reports have shown that pulmonary exposure to CNTs caused inflammation and lung fibrosis in rodents. The molecular mechanisms that govern CNT lung toxicity remain largely unaddressed. Here, we report that multiwalled carbon nanotubes (MWCNTs) have potent, dose-dependent toxicity on cultured human lung cells (BEAS-2B, A549, and WI38-VA13). Mechanistic analyses were carried out at subtoxic doses (≤<em>20</em> μg/mL, ≤ 24 h). MWCNTs induced substantial ROS production and mitochondrial damage, implicating oxidative stress in cellular damage by MWCNT. MWCNTs activated the NF-κB signaling pathway in macrophages (RAW264.7) to increase the secretion of a panel of cytokines and chemokines (TNFα, <em>IL</em>-1β, <em>IL</em>-6, <em>IL</em>-10, and MCP1) that promote inflammation. Activation of NF-κB involved rapid degradation of IκBα, nuclear accumulation of NF-κBp65, binding of NF-κB to specific DNA-binding sequences, and transactivation of target gene promoters. Finally, MWCNTs induced the production of profibrogenic growth factors TGFβ1 and PDGF from macrophages that function as paracrine signals to promote the transformation of lung fibroblasts (WI38-VA13) into myofibroblasts, a key step in the development of fibrosis. Our results revealed that MWCNTs elicit multiple and intertwining signaling events involving oxidative damage, inflammatory cytokine production, and myofibroblast transformation, which potentially underlie the toxicity and fibrosis in human lungs by MWCNTs.

Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis are chronic inflammatory disorders of gastrointestinal tract. Although the etiology is incompletely understood, initiation and aggravation of the inflammatory process seem to be due to a massive local mucosal immune response. Interleukin-10 (<em>IL</em>-10) is a regulatory cytokine which inhibits both antigen presentation and subsequent pro-inflammatory cytokine release, and it is proposed as a potent anti-inflammatory biological therapy in chronic IBD. Many methods of <em>IL</em>-10 as a treatment for IBD have been published. The new strategies of <em>IL</em>-10 treatment, including recombinant <em>IL</em>-10, the use of genetically modified bacteria, gelatine microsphere containing <em>IL</em>-10, adenoviral vectors encoding <em>IL</em>-10 and combining regulatory T cells are discussed in this review. The advantages and disadvantages of these <em>IL</em>-10 therapies are summarized. Although most results of recombinant <em>IL</em>-10 therapies are disappointing in clinical testing because of lacking efficacy or side effects, therapeutic strategies utilizing gene therapy may enhance mucosal delivery and increase therapeutic response. Novel <em>IL</em>-10-related cytokines, including <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24, <em>IL</em>-26, <em>IL</em>-28 and <em>IL</em>-29, are involved in regulation of inflammatory and immune responses. The use of <em>IL</em>-10 and <em>IL</em>-10-related cytokines will provide new insights into cell-based and gene-based treatment against IBD in near future.

<em>IL</em>-6 is a pleiotropic cytokine that plays a critical role in bone resorption. We describe two allelic variants in the <em>IL</em>-6 promoter, -572 and -174 G->>C, that alone and in combination influence <em>IL</em>-6 activity in vitro and in vivo. The association of <em>IL</em>-6 -572 genotypes and -572/-174 G->>C haplotypes with serum C-reactive protein (CRP), serum and urinary C-terminal cross-linking of type I collagen (a marker of bone resorption), and osteocalcin (a marker of bone formation) was investigated in a cohort of healthy postmenopausal women (n = 495; mean age +/- SD, 72 +/- 5.7 yr). Among <em>IL</em>-6 -572 genotypes, CRP was 37% higher (P = 0.02) and urinary C-terminal cross-linking of type I collagen was <em>20</em>% higher (P = 0.01) in the presence of the C allele, whereas serum osteocalcin was not different. <em>IL</em>-6 -572/-174 haplotypes (G/C, G/G, and C/G) were significantly associated with all biochemical markers, and additive effects of the two polymorphic loci were found. Thus, there was a significant increase in the level of CRP (up to +79%; P = 0.007) and bone resorption markers (up to +32%; P = 0.017) with a decreasing number (from four to one) of <em>IL</em>-6 protective alleles -572G and -174C. In addition, there was a trend for lower age-adjusted bone mineral density at the lumbar spine in subjects with less <em>IL</em>-6 protective alleles (up to -9.6%; P = 0.037; P = 0.08 after further adjustment for weight). In conclusion, we describe two functional polymorphisms in the <em>IL</em>-6 gene regulatory region associated with <em>IL</em>-6 activity in postmenopausal women, both systemically (CRP) and locally in bone. As such, <em>IL</em>-6 polymorphisms are able to influence the risk of osteoporosis as well as other chronic disorders involving <em>IL</em>-6 activity.

Individuals exposed to inhaled endotoxin (lipopolysaccharide [LPS]) can develop airway symptomatology and exacerbations of asthma. Moreover, among those occupationally exposed to organic dusts, the progression of airflow obstruction is related to the endotoxin concentration in the bioaerosol. Not everyone exposed to high concentrations of LPS develops these problems. To determine whether individuals express a differential response to inhaled LPS, we challenged 72 healthy volunteers with increasing doses of LPS. Airflow was assessed after each dose and the protocol was terminated for decline in FEV1>>/= <em>20</em>%. Marked differences in the response to inhaled LPS were observed: eight "sensitive" subjects had at least <em>20</em>% decline in their FEV1 after inhaling 6.5 micrograms or less of LPS, whereas 11 "hyporesponsive" subjects maintained an FEV1>>/= 90% of their baseline even after inhaling 41.5 micrograms of LPS. Serial testing demonstrated that the response to inhaled LPS is reproducible. Sensitive subjects were more commonly female and hyporesponsive subjects were more often male (p = 0.016). Peripheral blood monocytes from hyporesponsive subjects, compared with sensitive subjects, released less interleukin (<em>IL</em>)-6 and <em>IL</em>-8. These findings demonstrate that an LPS phenotype can be reproducibly elicited in humans, which creates an opportunity to identify genes involved in this response to inhaled LPS.

OBJECTIVE

To investigate cytokine gene expression in the lung after single and fractionated doses of radiation, and to investigate the effect of steroids and the genetic background.

METHODS

Expression of cytokine genes (mTNF-alpha, mIL-1alpha, mIL-1beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-gamma) in the lungs of C3H/HeJ and C57BL/6J mice was measured by RNase protection assay at different times after various doses of radiation. The effects of dexamethasone and fractionated radiation treatment on gene expression were also studied.

RESULTS

<em>IL</em>-1beta was the major cytokine induced in the lungs of C3H/HeJ mice within the first day after thoracic irradiation. Radiation doses as low as 1 Gy were effective. Responses to <em>20</em> Gy irradiation peaked within 4-8h and subsided by 24 h. With the exception of <em>IL</em>-1alpha and TNF-alpha, the other cytokines that were investigated had undetectable pre-treatment mRNA levels and were not radiation inducible. Similar responses were seen in C57BL/6J mice, although TNF-alpha was induced and there were some quantitative differences. Pre-treatment of C3H/HeJ mice with dexamethasone reduced basal and induced <em>IL</em>-1 levels, but complete inhibition was not achieved. Dexamethasone was also effective if given immediately after irradiation. Fractionated daily doses of radiation (4 Gy/day) helped to maintain cytokine gene expression for a longer period.

CONCLUSIONS

Inflammatory genes are rapidly induced in the lung by irradiation. This response cannot be readily abolished by steroid pre-treatment. Fractionated treatment schedules help to perpetuate the response.

Eosinophils, through their ability to generate an array of potent mediators, are thought to be the major effector cells in a number of conditions, including parasitic infection, asthma, and other allergic diseases. The mechanism(s) by which eosinophils, as opposed to neutrophils, accumulate at inflammatory sites is unknown. One possible mechanism would be an eosinophil-specific pathway of adhesion to vascular endothelium. In this study we have demonstrated that human eosinophils, but not neutrophils, constitutively express alpha 4 beta 1 (CD49d/CD29). Expression was not increased on low density eosinophils or normal density cells stimulated with platelet-activating factor. Eosinophils, but not neutrophils, specifically adhered to COS cells transfected with vascular adhesion molecule-1 in a alpha 4 beta 1-dependent manner. Eosinophil, but not neutrophil, adhesion to <em>IL</em>-1 stimulated human umbilical vascular endothelial cells was significantly inhibited by alpha 4 beta 1 mAb at both 5 h (p less than 0.05) and <em>20</em> h (p less than 0.001). Inhibition of both resting and platelet-activating factor-(10(-7) M) stimulated eosinophil adhesion was observed. We conclude that the alpha 4 beta 1/vascular adhesion molecule-1 adhesion pathway may be involved in specific eosinophil, as opposed to neutrophil, migration into sites of eosinophilic inflammation.

The urine of febrile patients has been found to contain high concentrations of an inhibitor of interleukin 1 (<em>IL</em>-1)-induced thymocyte proliferation. The inhibitor is specific for <em>IL</em>-1 and does not block the effects of interleukin 2 (<em>IL</em>-2) or phytohemagglutin (PHA) on thymocytes, and it is not nonspecifically toxic for these cells. <em>IL</em>-1 inhibitor can be found in the urine of normal individuals and afebrile patients, but is present in increased concentrations in the urine of patients with fever of diverse etiologies. Preliminary physicochemical characterization indicates that the inhibitor is a <em>20</em>-40-kdalton protein.

Five novel cytokines (<em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22 (<em>IL</em>-TIF), <em>IL</em>-24 (human MDA-7, mouse FISP, rat C49A/Mob-5), and <em>IL</em>-26 (AK155)) demonstrating limited primary sequence identity and probable structural homology to <em>IL</em>-10 have been identified. These cellular cytokines, as well as several cytokines encoded in viral genomes (viral cytokines), form a family of <em>IL</em>-10-related cytokines or the <em>IL</em>-10 family. These cytokines share not only homology but also receptor subunits and perhaps activities. Receptors for these cytokines belong to the class II cytokine receptor family. The receptors are <em>IL</em>-10R2 (CRF2-4), <em>IL</em>-22R1 (CRF2-9), <em>IL</em>-22BP (CRF2-10), <em>IL</em>-<em>20</em>R1 (CRF2-8) and <em>IL</em>-<em>20</em>R2 (CRF2-11). Biological activities of these cytokines, receptor utilization and signaling, as well as expression patterns for cytokines and their receptors are summarized. Although data indicate that these cytokines are involved in regulation of inflammatory and immune responses, their major functions remain to be discovered.

The properties of specific human interleukin 1 (<em>IL</em> 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human <em>IL</em> 1-beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton-Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-<em>IL</em> 1-beta. Maximal binding was reached within <em>20</em> min at 4 degrees C. Scatchard plot analysis of the binding of 125I-<em>IL</em> 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 2<em>20</em> binding (receptor) sites/cell. The binding of 125I-<em>IL</em> 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human <em>IL</em> 1 and recombinant human <em>IL</em> 1-alpha, as well as by unlabeled human <em>IL</em> 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that <em>IL</em> 1-alpha and <em>IL</em> 1-beta bind specifically to the same receptor. The m.w. of <em>IL</em> 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human <em>IL</em> 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of <em>IL</em> 1 receptor on human EBV-B cells additionally supports the hypothesis that <em>IL</em> 1 may be an autocrine signal for these cells.