NF-kappaB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF-kappaB from IkappaBalpha and degradation of IkappaBalpha, targeting for degradation and signaling are different. Early IkappaBalpha degradation at 30 min to approximately 6 h is not initiated by UV-induced DNA damage. It does not require IkappaB kinase (IKK), as shown by introduction of a dominant-negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IkappaBalpha, carrying serines at positions 32 and 36. Induced IkappaBalpha degradation requires, however, intact N- (positions 1-36) and C-terminal (positions 277-287) sequences. IkappaB degradation and NF-kappaB activation at late time points, 15-<em>20</em> h after UV irradiation, is mediated through DNA damage-induced cleavage of <em>IL</em>-1alpha precursor, release of <em>IL</em>-1alpha and autocrine/paracrine action of <em>IL</em>-1alpha. Late-induced IkappaBalpha requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the 'alarmone' <em>IL</em>-1alpha may account for some of the systemic effects of sunlight exposure.

Human polymorphonuclear leukocytes (PMN) stimulated by lipopolysaccharide (LPS) produce interleukin-12 (<em>IL</em>-12). Both the free <em>IL</em>-12 p40 chain and minute amounts of the biologically active <em>IL</em>-12 p70 heterodimers are produced by PMN. Interferon-gamma (IFN-gamma) enhanced the LPS-induced secretion of both the free <em>IL</em>-12 p40 chain and the p70 heterodimer by approximately fivefold. As observed for other <em>IL</em>-12-producing cell types, the ratio of free p40 chain to p70 heterodimer secreted by LPS-stimulated PMN was approximately <em>20</em>:1. LPS induced a 100-fold increase of <em>IL</em>-12 p40 mRNA, but had minimal effect on p35 mRNA accumulation. IFN-gamma enhanced the LPS-induced accumulation of p40 mRNA and directly induced a several-fold increase in the accumulation of p35 mRNA. Therefore, the combined effect of LPS and IFN-gamma induced sufficient expression of both p40 and p35 to attain production of the biologically active p70 heterodimer at physiologically relevant concentrations. The ratio between p40 and p35 mRNA abundance in PMN stimulated with both LPS and IFN-gamma was approximately <em>20</em>0:1, explaining the secretion of the free p40 chain in much higher concentrations than the p70 heterodimer. <em>IL</em>-10, an inhibitor of the production of various cytokines in PMN, also suppressed <em>IL</em>-12 mRNA accumulation and secretion by PMN. Because of the important immunoregulatory function of <em>IL</em>-12, in particular induction of IFN-gamma production and facilitation of T helper cell type 1 response, the ability of PMN to produce <em>IL</em>-12 suggests that neutrophils may play an active role in the regulatory interaction between innate resistance and adaptive immunity.

A deletion of about <em>20</em> amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (<em>IL</em>-6), transforming growth factor-beta4 (TGF-beta4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.

We have used <em>IL</em>-10 gene knockout mice (<em>IL</em>-10T) to examine the role of endogenous <em>IL</em>-10 in the down-modulation of hepatic granuloma formation and lymphocyte responses that occurs in chronic infection with the helminth parasite Schistosoma mansoni. Although <em>IL</em>-10-deficient animals showed <em>20</em> to 30% mortality between 8 and 14 wk postinfection, they displayed no alterations in their susceptibility to infection and produced similar numbers of eggs as their wild-type littermates. The <em>IL</em>-10T mice displayed a significant increase in hepatic granuloma size at the acute stage of infection, which was associated with increased IFN-gamma, <em>IL</em>-2, <em>IL</em>-1beta, and TNF-alpha mRNA expression in liver and elevated Th1-type cytokine production by lymphoid cells. Despite developing an enhanced Th1-type cytokine response, the <em>IL</em>-10T mice showed no consistent decrease in their Th2-type cytokine profile. Surprisingly, although granulomatous inflammation was enhanced at the acute stage of infection, the livers of <em>IL</em>-10T mice displayed no significant increase in fibrosis and underwent normal immune down-modulation at the chronic stage of infection. Moreover, the down-modulated state could be induced in <em>IL</em>-10T mice by sensitizing the animals to schistosome eggs before infection, further demonstrating that the major down-regulatory mechanism is not dependent upon <em>IL</em>-10. We conclude that while <em>IL</em>-10 plays an important role in controlling acute granulomatous inflammation, it plays no essential role in the process of immune down-modulation in chronic schistosome infection.

Ag-specific immune tolerance results from the induction of cellular mechanisms that limit T cell responses to selective Ags. One of these mechanisms is characterized by attenuated proliferation and decreased <em>IL</em>-2 production in fully stimulated CD4(+) Th cells and is denoted T cell anergy. We report the identification of the early growth response gene (Egr-2; Krox-<em>20</em>), a zinc-finger transcription factor, as a key protein required for induction of anergy in cultured T cells. Gene array screening revealed high Egr-2 expression distinctly persists in anergized but not proliferating murine A.E7 T cells. In contrast, Egr-1, a related family member induced upon costimulation, displays little or no expression in the anergic state. <em>IL</em>-2-mediated abrogation of anergy causes rapid depletion of Egr-2 protein. Full stimulation of anergic A.E7 T cells fails to enhance <em>IL</em>-2 and Egr-1 expression, whereas Egr-2 expression is greatly increased. Silencing Egr-2 gene expression by small interfering RNA treatment of cultured A.E7 T cells before incubation with anti-CD3 alone prevents full induction of anergy. However, small interfering RNA-mediated depletion of Egr-2 5 days after anergy induction does not appear to abrogate hyporesponsiveness to stimulation. These data indicate that sustained Egr-2 expression is necessary to induce a full anergic state through the actions of genes regulated by this transcription factor.

Shigella infection is accompanied by an intestinal activation of epithelial cells, T cells, and macrophages within the inflamed colonic mucosa. A prospective study was carried out to elucidate the cytokine pattern in Shigella infection linked to development of immunity and eradication of bacteria from the local site and also to correlate the cytokine profile with histological severity. An indirect immunohistochemical technique was used to determine the production and localization of various cytokines at the single-cell level in cryopreserved rectal biopsies from 24 patients with either Shigella dysenteriae type 1 (n = 18) or Shigella flexneri (n = 6) infection. The histopathological profile included presence of chronic inflammatory cells with or without neutrophils and microulcers in the lamina propria, crypt distortion, branching, and less frequently crypt abscesses. Patients had significantly higher (P < 0.005) numbers of cytokine producing cells for all of the cytokines studied, interleukin-1 alpha (<em>IL</em>-1 alpha), <em>IL</em>-1 beta, <em>IL</em>-1ra, tumor necrosis factor alpha (TNF-alpha), <em>IL</em>-6, <em>IL</em>-8, <em>IL</em>-4, <em>IL</em>-10, gamma interferon, TNF-beta, and transforming growth factor beta 1-3, in the biopsies than the healthy controls (n = 13). The cytokine production profile during the study period was dominated by <em>IL</em>-1 beta, transforming growth factor beta 1-3, <em>IL</em>-4, and <em>IL</em>-10. Significantly increased frequencies of cytokine-producing cells (P < 0.05) were observed for <em>IL</em>-1, <em>IL</em>-6, gamma interferon, and TNF-alpha in biopsies with severe inflammation in comparison with those with mild inflammation. During the acute stage of the disease, <em>20</em> of 24 patients exhibited acute inflammation in the rectal biopsies and the cellular infiltration was still extensive 30 days after the onset of diarrhea, although the disease was clinically resolved. In accordance with the histological findings, cytokine production was also upregulated during the convalescent phase; there was no significant difference (P>> 0.05) in the incidence of cytokine-producing cells between acute (2 to 8 days after the onset of diarrhea) and convalescent (30 days after onset) stages.

Antenatal exposure to glucocorticoids, amnionitis, intraamniotic interleukin (<em>IL</em>)-1alpha, or endotoxin can improve postnatal lung function after preterm delivery. The relationship between early lung maturation and the dose and duration of a proinflammatory stimulus has not been evaluated. The effects of proinflammatory stimuli on fetal plasma cortisol also have not been evaluated. We hypothesized that intraamniotic endotoxin would induce early lung maturation in fetal sheep without increasing fetal cortisol. Intraamniotic injections of 1, 4, <em>20</em>, or 100 mg of Escherichia coli 055:beta5 endotoxin caused 2-fold increases in compliance, 4- to 5-fold increases in lung gas volumes, and <em>20</em>-fold increases in alveolar saturated phosphatidylcholine (Sat PC) when given 7 d before preterm delivery at 125 d gestation. Animals treated with <em>20</em> mg endotoxin for treatment to delivery intervals of 5 h to 15 d had no significant elevations in cord plasma cortisol levels. Increases in Sat PC in lung tissue and alveolar washes were detected 2 d after endotoxin treatment and lung function improved 4 d after endotoxin treatment. Two doses of endotoxin given 3 and 7 d or 7 and 15 d before treatment resulted in lung maturation responses equivalent to single dose comparison groups without elevations in cortisol. Early lung maturation induced by intraamniotic endotoxin in fetal sheep occurred without an increase in fetal plasma cortisol, indicating that endotoxin promoted lung maturation by a mechanism independent of cortisol.

OBJECTIVE

Inflammatory reaction has been shown to involve the progress of type 2 (non-insulin-dependent) diabetes. We, therefore, examined the effects of inflammatory cytokines and angiogenic factors in the pathogenesis of proliferative diabetic retinopathy (PDR) in type 2 diabetes.

METHODS

Vitreous fluid samples were obtained by vitrectomy from 62 eyes of PDR patients with type 2 diabetes and from <em>20</em> eyes of age-matched non-diabetic patients. The concentrations of interleukin 1 beta (IL1B), IL6, IL8, IL10, chemokine (C-C motif) ligand 2 (CCL2), endothelin 1 (EDN1), vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF) in the vitreous samples were measured by enzyme-linked immunosorbent assay (ELISA).

RESULTS

The concentrations of LI1B, IL6, IL8, CCL2, EDN1, VEGF, and TNF in the vitreous samples were considerably higher in PDR patients in comparison with the controls. However, the level of IL10 in PDR patients was similar to that obtained in the controls. Analysis of the correlations of the studied factors revealed the correlation of VEGF and IL6, VEGF and EDN1, IL8 and CCL2, and EDN1 and TNF in PDR patients. In addition, a significant positive correlation was observed between vitreous TNF as well as EDN1 and serum HbA(1)c levels in PDR patients.

CONCLUSIONS

The inflammatory cytokines and angiogenic factors IL1B, IL6, IL8, CCL2, EDN1, VEGF, and TNF are increased in the vitreous of PDR patients without an increase in IL-10. These results add support to the role of inflammatory cytokines and angiogenic factors in the genesis of PDR. Understanding the implication of these cytokines may provide diagnostic tools and therapeutic targets for treatment and prevention of PDR.

OBJECTIVE

To test the hypotheses that obesity due to a very high-fat diet induces knee osteoarthritis (OA), and that short-term wheel-running exercise protects against obesity-induced knee OA by reducing systemic inflammation and metabolic dysregulation.

METHODS

Male C57BL/6J mice were fed either a control diet (13.5% kcal from fat) or a very high-fat diet (60% kcal from fat) from age 12 weeks to age 24 weeks. From <em>20</em> to 24 weeks of age, half of the mice were housed with running wheels. The severity of knee OA was determined by assessing histopathologic features, and serum cytokines were measured using a multiplex bead immunoassay and enzyme-linked immunosorbent assays. Body composition was quantified by dual-energy x-ray absorptiometry, and insulin resistance was assessed by glucose tolerance testing.

RESULTS

Feeding mice with a very high-fat diet increased knee OA scores and levels of serum leptin, adiponectin, KC (mouse analog of interleukin-8 [IL-8]), monokine induced by interferon-γ (CXCL9), and IL-1 receptor antagonist to an extent in proportion to the gain in body fat (3-fold increase in percent body fat compared to controls). Wheel-running exercise reduced progression of OA in the medial femur of obese mice. In addition, exercise disrupted the clustering of cytokine expression and improved glucose tolerance, without reducing body fat or cytokine levels.

CONCLUSIONS

Obesity induced by a very high-fat diet in mice causes OA and systemic inflammation in proportion to body fat. Increased joint loading is not sufficient to explain the increased incidence of knee OA with obesity, as wheel running is protective rather than damaging. Exercise improves glucose tolerance and disrupts the coexpression of proinflammatory cytokines, suggesting that increased aerobic exercise may act independently of weight loss in promoting joint health.

Atopic dermatitis (AD) can be categorized into the extrinsic and intrinsic types. Extrinsic or allergic AD shows high total serum IgE levels and the presence of specific IgE for environmental and food allergens, whereas intrinsic or non-allergic AD exhibits normal total IgE values and the absence of specific IgE. While extrinsic AD is the classical type with high prevalence, the incidence of intrinsic AD is approximately <em>20</em>% with female predominance. The clinical features of intrinsic AD include relative late onset, milder severity, and Dennie-Morgan folds, but no ichthyosis vulgris or palmar hyperlinearity. The skin barrier is perturbed in the extrinsic, but not intrinsic type. Filaggrin gene mutations are not a feature of intrinsic AD. The intrinsic type is immunologically characterized by the lower expression of interleukin (<em>IL</em>) -4, <em>IL</em>-5, and <em>IL</em>-13, and the higher expression of interferon-gamma. It is suggested that intrinsic AD patients are not sensitized with protein allergens, which induce Th2 responses, but with other antigens, and metals might be one of the candidates of such antigens.

<em>IL</em>-10 plays an important role in the regulation of immune responses. We and others have demonstrated recently that cyclic adenosine monophosphate (cAMP)-elevating substances up-regulate monocytic <em>IL</em>-10 expression in vitro and in vivo. Computer analysis of the <em>IL</em>-10 promoter/enhancer region localized four putative cAMP-responsive elements (CRE1- 4) with homology to the CRE consensus motif. In electrophoretic mobility shift assays CRE1 and CRE4 bound protein complexes consisting of transcription factors CREB-1 and ATF-1, while CRE3 bound only marginal amounts of CREB-1/ATF-1 in combination with unknown protein(s). CRE2 showed no protein binding activity. In vitro mutation of CRE1 and CRE4 reduced the level of cAMP-stimulated transactivation in reporter gene assays in comparison to the wild-type promoter by <em>20</em> % and 50 %, respectively, while mutation of CRE3 had no effect. The main action of CRE4 on cAMP-dependent stimulation is probably based on its adjacent localization to the TATA box and its sequence comprising a perfect half site. Experiments with double and triple mutants and with deleted promoter fragments indicated the participation of additional elements beside the CRE motifs in the cAMP-dependent stimulation. Our data suggest that intracellular cAMP may directly affect expression of the immunoregulatory cytokine <em>IL</em>-10 in monocytic cells via activation of the eukaryotic transcription factors CREB-1 and ATF-1 and their binding to CRE1 and CRE4 in the upstream enhancer of the <em>IL</em>-10 promoter.

The aim of this study was to determine the release and regulation of leptin, resistin and adiponectin from human placenta and fetal membranes, and maternal subcutaneous adipose tissue and skeletal muscle obtained from normal and gestational diabetes mellitus (GDM)-complicated pregnancies at the time of Cesarean section. Tissue explants were incubated in the absence (basal control) or presence of 10 mug/ml lipopolysaccharide (LPS), 10, <em>20</em> or 40 ng/ml tumor necrosis factor-alpha (TNF-alpha), interleukin (<em>IL</em>)-6 and <em>IL</em>-8, 1 microM phorbol myristate acetate, 10, <em>20</em> and 40 mM glucose, 0.1, 1 and 10 microM insulin and 0.1 1 and 10 microM dexamethasone, progesterone and estrogen. After an 18-h incubation, the medium was collected and the release of leptin, resistin and adiponectin was quantified by ELISA. Human gestational tissues and maternal tissues released immunoreactive leptin, resistin and adiponectin; however, there was no difference in the release of either resistin or adiponectin between normal pregnant women and women with gestational diabetes. The release of leptin was significantly higher in placenta, amnion and choriodecidua obtained from normal pregnant women compared with women with GDM. However, in maternal tissues, the situation was reversed, with adipose tissue and skeletal muscle obtained from women with GDM releasing significantly greater amounts of leptin. In adipose tissue and skeletal muscle the release of leptin was significantly greater in insulin-controlled GDM compared with diet-controlled GDM, and leptin release from adipose tissue was significantly correlated with maternal body mass index. In all tissues tested, there was no effect of incubation with LPS, <em>IL</em>-6, <em>IL</em>-8 or TNF-alpha on leptin, resistin or adiponectin release. PMA significantly increased the release of resistin from placenta and adipose tissue. Insulin increased placental resistin release, whereas the hormones dexamethasone, progesterone and estrogen significantly decreased placental resistin release. The data presented in this study demonstrate that dysregulation of leptin metabolism and/or function in the placenta may be implicated in the pathogenesis of GDM. Furthermore, resistin release is greatly affected by a variety of inflammatory mediators and hormones.

OBJECTIVE

We report survival, prognostic factors, and treatment efficacy in low-grade glioma.

METHODS

A total of 379 patients with histologic intracranial low-grade glioma received post-operative radiotherapy (n = 361) and intraarterial carmustine (BCNU) chemotherapy (n = 153). Overall survival and prognostic factors were evaluated with the SPSS statistical program (SPSS Inc, Chicago, IL).

RESULTS

Median survival (all patients) was 100 months (95% confidence interval [CI], B7 to 113); in age group 0 to 19 years (n = 41), 226 months; in age group 20 to 49 years (n = 263), 106 months; in age group 50 to 59 years (n = 49), 76 months; and for older patients (n = 26), 39 months. Projected survival at 10 and 15 years was 42% and 29%, respectively. Patient age, World Health Organization (WHO) performance status, tumor computed tomography (CT) contrast enhancement, mental changes, or initial corticosteroid dependency were significant independent prognostic factors (p < .05), while histologic subgroup, focal deficits, presence of seizures, prediagnostic symptom duration, tumor category, and tumor stage were not. Patients aged 20 to 49 years with no independent negative prognostic factors (n = 132) had a median survival time of 139 months versus 41 months in patients with two or more factors (n = 33). Patients who presented with symptoms of expansion (n = 97) survived longer when resected (P < .03); otherwise no survival benefit was associated with initial tumor resection compared with biopsy. Intraarterial chemotherapy and radiation doses more than 55 Gy were not associated with prolonged survival. Among 66 reoperated patients, 45% progressed to high-grade histology within 25 months.

CONCLUSIONS

Prognosis in low-grade glioma following postoperative radiotherapy seems largely determined by the inherent biology of the glioma and patient age at diagnosis.

OBJECTIVE

We sought to investigate the effects of physical training on circulating proinflammatory cytokines and the soluble apoptosis mediators Fas (sFas) and Fas ligand (sFasL) in patients with chronic heart failure (CHF).

BACKGROUND

Recent investigations have shown an overexpression of circulating proinflammatory cytokines and soluble apoptosis mediators in patients with CHF, which may be related to their exercise intolerance and clinical deterioration.

METHODS

Plasma levels of tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptors I and II (sTNF-RI and sTNF-RII, respectively), interleukin-6 (<em>IL</em>-6), soluble <em>IL</em>-6 receptor (s<em>IL</em>-6R), sFas and sFasL were measured in 24 patients with stable CHF (New York Heart Association functional class II/III; left ventricular ejection fraction 23.2 +/- 1.3%) and in <em>20</em> normal control subjects before and after a 12-week program of physical training in a randomized, crossover design. Functional status of patients with CHF was evaluated by using a cardiorespiratory exercise test to measure peak oxygen consumption (VO2max).

RESULTS

Physical training produced a significant reduction in plasma levels of TNF-alpha (7.5 +/- 1.0 pg/ml vs. 4.6 +/- 0.7 pg/ml, p < 0.001), sTNF-RI (3.3 +/- 0.2 ng/ml vs. 2.7 +/- 0.2 ng/ml, p < 0.005), sTNF-RII (2.6 +/- 0.2 ng/ml vs. 2.3 +/- 0.2 ng/ml, p = 0.06), IL-6 (8.3 +/- 1.2 pg/ml vs. 5.9 +/- 0.8 pg/ml, p < 0.005), sIL-6R (34.0 +/- 3.0 ng/ml vs. 29.2 +/- 3.0 ng/ml, p < 0.01), sFas (5.5 +/- 0.7 ng/ml vs. 4.5 +/- 0.8 ng/ml, p = 0.05) and sFasL (34.9 +/- 5.0 pg/ml vs. 25.2 +/- 4.0 pg/ml, p < 0.05), as well as a significant increase in VO2max (16.3 +/- 0.7 ml/kg per min vs. 18.7 +/- 0.8 ml/kg per min, p < 0.001). Good correlations were found between a training-induced increase in VO2max and a training-induced reduction in levels of the proinflammatory cytokine TNF-alpha (r = -0.54, p < 0.01) and the apoptosis inducer sFasL (r = -0.57, p < 0.005) in patients with CHF. In contrast, no significant difference in circulating cytokines and apoptotic markers was found with physical training in normal subjects.

CONCLUSIONS

Physical training reduces plasma levels of proinflammatory cytokines and the sFas/sFasL system in patients with CHF. These immunomodulatory effects may be related to the training-induced improvement in functional status of patients with CHF.

BACKGROUND

IL-1beta is a proinflammatory cytokine driving joint inflammation as well as systemic signs of inflammation, such as fever and acute phase protein production.

METHODS

ACZ885, a fully human monoclonal antibody that neutralizes the bioactivity of human IL-1beta, was generated to study the potent and long-lasting neutralization of IL-1beta in mechanistic animal models as well as in a proof-of-concept study in patients with rheumatoid arthritis (RA).

RESULTS

The mouse IL-1 receptor cross-reacts with human IL-1beta, and it was demonstrated that ACZ885 can completely suppress IL-1beta-mediated joint inflammation and cartilage destruction in mice. This observation prompted us to study the safety, tolerability and pharmacodynamic activity of ACZ885 in RA patients in a small proof-of-concept study--the first to be conducted in humans. Patients with active RA despite treatment with stable doses of methotrexate were enrolled in this dose escalation study. The first 32 patients were split into four cohorts of eight patients each (six were randomly assigned to active treatment and two to placebo). ACZ885 doses were 0.3, 1, 3 and 10 mg/kg, administered intravenously on days 1 and 15. To explore efficacy within 6 weeks of treatment, an additional 21 patients were randomly assigned to the 10 mg/kg cohort, resulting in a total of 20 patients dosed with 10 mg/kg and 15 patients treated with placebo. There was clinical improvement (American College of Rheumatology 20% improvement criteria) at week 6 in the 10 mg/kg treatment group; however, this did not reach statistical significance (P = 0.085). A statistically significant reduction in disease activity score was observed after 4 weeks in the 10 mg/kg group. Onset of action was rapid, because most responders exhibited improvement in their symptoms within the first 3 weeks. C-reactive protein levels decreased in patients treated with ACZ885 within 1 week. ACZ885 was well tolerated. Three patients receiving ACZ885 developed infectious episodes that required treatment. No anti-ACZ885 antibodies were detected during the study.

CONCLUSIONS

ACZ885 administration to methotrexate-refractory patients resulted in clinical improvement in a subset of patients. Additional studies to characterize efficacy in RA and to determine the optimal dose regimen appear warranted.

BACKGROUND

ClinicalTrials.gov identifier NCT00619905.

To explore the activation patterns of signal transducer and activator of transcription 3 (Stat3) in acute myeloid leukemia (AML), we examined whether the phosphorylation of tyrosine705 (Tyr705) and serine727 (Ser727) residues was abnormally regulated in cells from patients with AML. In 5 of <em>20</em> (25%) patients with AML, Stat3 was constitutively phosphorylated on Tyr705 and Ser727, which were not further up-regulated by treatment with <em>IL</em>-6. Furthermore, Stat3 was constitutively bound to the IRE response element in these cells as determined by electrophoretic mobility shift assay, and stimulation with <em>IL</em>-6 did not result in increased DNA binding. Interestingly, AML cells with constitutive Stat3 activation also secreted high levels of <em>IL</em>-6 protein. Treating these AML cells with anti-<em>IL</em>-6 resulted in restored <em>IL</em>-6-inducible Stat3 phosphorylation on both Tyr705 and Ser727 with low or undetectable basal phosphorylation levels in unstimulated cells. In contrast, treatment with anti-<em>IL</em>-1 did not result in altered Stat3 phosphorylation patterns. The constitutive <em>IL</em>-6 expression was associated with elevated levels of suppressor of cytokine signaling-1 (SOCS-1) and SOCS-3 mRNA expression, which were not down-regulated by anti-<em>IL</em>-6. These data indicate that the constitutive Stat3 activation in the investigated AML blasts is caused by high <em>IL</em>-6 secretion levels, thus stimulating the Jak/Stat pathway in an autocrine manner, a paracrine manner, or both. (Blood. <em>20</em>00;95:3765-3770)

BACKGROUND

Leishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis.

METHODS

To explore immunoregulatory mechanisms, <em>20</em> visceral leishmaniasis (VL) patients were evaluated during active disease and at different periods up to one year after treatment determining their plasma cytokine levels, clinical parameters (palpable spleen and liver) and antibody levels.

RESULTS

Elevated plasma levels of IFN-gamma and of IL-12 p40 were observed during active disease, significantly decreasing after treatment whereas in vitro Leishmania antigen-stimulated IFN-gamma production by PBMC exhibited an inverse pattern being low during disease and increasing steadily thereafter. Absence of IFN-gamma activity is a hallmark of VL. The main candidate for blunting IFN-gamma activity is IL-10, a cytokine highly elevated in plasma with sharp decrease after treatment. Activity of IL-10 is inferred by high levels of anti-Leishmania specific IgG1 and IgG3. TGF-beta had elevated total, but not of active, levels lessening the likelihood of being the IFN-gamma counterpart. Spleen or liver size presented a steady decrease but return to normal values at only 1<em>20</em> days after treatment. Anti-Leishmania IgG (total and subclasses) levels and DTH or Leishmania-stimulated lymphocyte proliferation conversion to positive also present a slow decrease after treatment. IL-6 plasma levels were elevated in only a few patients.

CONCLUSIONS

Taken together our results suggest that IFN-gamma and IL-10 are the molecules most likely involved in determining fate of disease. After treatment, there is a long delay before the immune profile returns to normal what precludes using plasma cytokine levels as criteria of cure as simpler clinical evaluations, as a palpable spleen or liver, can be used.

The aim of the study was to investigate whether interleukin-10 (<em>IL</em>-10) genetic polymorphisms influence this cytokine production as well as the incidence and outcome of diffuse large B-cell lymphoma (DLBCL). The frequency of <em>IL</em>-10(-1082G) allele was found to be higher in 199 patients with DLBCL as compared with 112 control subjects (0.47 versus 0.39, P =.043). Increased serum levels of <em>IL</em>-10 were associated with adverse prognostic factors and poor DLBCL outcome. The frequencies of <em>IL</em>-10(-819T) and <em>IL</em>-10(-592A) alleles were lower in patients with elevated <em>IL</em>-10 serum levels (0.155 versus 0.32, P =.14). As compared with patients carrying the <em>IL</em>-10(-1082AA) genotype, patients with the <em>IL</em>-10(-1082G) allele (<em>IL</em>-10(-1082GG/GA) genotypes) had higher complete remission rate (78% [confidence interval (CI), 71%-85%] versus 65% [CI, 52%-78%], P =.07), 5-year freedom from progression (FFP) (60% [CI, 52%-68%] versus 40% [CI, 27%-53%], P =.013), and overall survival (OS) (63% [CI, 55%-71%] versus 33% [CI, <em>20</em>%-45%], P =.0009). Among factors of the International Prognostic Index, <em>IL</em>-10(-1082G) allele remained an independent variable, predicting longer freedom from progression (FFP) (RR [relative risk] =.76, P =.00035) and OS (RR =.78, P =.0015). These results indicate that <em>IL</em>-10 production contributes to the clinical course of DLBCL and that this phenomenon involves a substantial genetic component.

We have previously shown that orally administered myelin basic protein (MBP) suppresses experimental autoimmune encephalomyelitis in both the Lewis rat and the SJL mouse. In the Lewis rat fed low doses of MBP, we found that protection can be adoptively transferred by CD8+ cells and that these cells inhibit immune responses via the secretion of TGF-beta after Ag-specific triggering. In the present study, we investigated the cellular requirements for the generation of active suppression following oral administration of MBP in SJL and (PLJ x SJL)F1 mice. We first determined the frequency of MBP cells secreting Th1 (IFN-gamma) and Th2 (<em>IL</em>-4/<em>IL</em>-10) cytokines or TGF-beta after oral administration of MBP. We found that in SJL mice, orally administered MBP (0.5 mg/feeding) led to an increased frequency of TGF-beta-, <em>IL</em>-4-, and <em>IL</em>-10-secreting cells and a decreased frequency of IFN-gamma-producing cells. This pattern was observed in both CD4+ and CD8+ populations; adoptive transfer of either CD4+ or CD8+ cells from orally tolerized mice suppressed autoimmune encephalomyelitis in recipient animals. We then studied the role of CD8+ cells on the generation of oral tolerance to MBP by depleting CD8+ cells in vivo with anti-CD8 mAb. Oral tolerance was successfully induced in such animals, as demonstrated by a decrease in clinical disease and T cell proliferative responses, although there was less TGF-beta production in vitro and less disease protection on days <em>20</em> to 22 in CD8-depleted animals. These studies demonstrate that CD4+ cells in the absence of CD8+ cells can mediate the active suppression component of oral tolerance in mice and that there is a reciprocal relationship between Th1- and Th2-type cytokine production associated with oral tolerization.

Resistance to murine leishmaniasis correlates with development of a CD4(+) T helper 1 (Th1)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN), known to promote a Th1 immune response, could provide protection from Leishmania infection, CpG-ODN and freeze-thawed (F/T) Leishmania major were coinjected intradermally into susceptible BALB/c mice. A Leishmania-specific Th1-predominant immune response was induced, and 40% of animals were protected from subsequent challenge with infectious organisms, with 0% protection of animals injected with F/T Leishmania organisms and PBS, F/T organisms and control ODN, or F/T organisms alone. More striking protection (65-95%) was seen in mice first infected with intact Leishmania organisms and then injected with CpG-ODN, either at the site of infection or at a remote site. To determine whether the therapeutic protection provided by CpG-ODN depended on <em>IL</em>-12 and IFN-gamma production, both IFN-gamma-deficient BALB/c mice and BALB/c mice treated with neutralizing anti-<em>IL</em>-12 mAb were first inoculated with Leishmania and then treated with either CpG-ODN, ODN, or PBS. None of these IFN-gamma-deficient mice survived (0/<em>20</em>, 0/<em>20</em>, and 0/<em>20</em> respectively). Furthermore, neutralization of <em>IL</em>-12 completely abolished the therapeutic protection provided by CpG-ODN (0/<em>20</em> mice surviving). We conclude that immunostimulatory DNA sequences likely exert systemic effects via <em>IL</em>-12 and IFN-gamma-dependent mechanisms and hold considerable promise as both vaccine adjuvants and potential therapeutic agents in the prevention and treatment of leishmaniasis.

BACKGROUND

Interleukin-12 (IL-12) is a macrophage-derived cytokine that modulates T lymphocyte responses and has the capacity to suppress allergic and eosinophilic inflammation.

METHODS

We carried out a double-blind, randomised, parallel group clinical study, in which patients with mild allergic asthma were given subcutaneous recombinant human IL-12 at increasing weekly injections of 0.1, 0.25, 0.5 microg/kg (n=19), or placebo (n=20). We compared responses to inhaled allergen challenge 24 h before the first injection and 24 h after the final injection. Airways hyper-responsiveness and concentrations of peripheral blood eosinophils and sputum eosinophils were also assessed.

RESULTS

IL-12 caused a significant decrease from baseline in the main peripheral blood eosinophil count 24 h after the fourth injection compared with placebo (p=0.0001). Sputum eosinophils were also significantly decreased 24 h after allergen challenge when treated with IL-12 compared with placebo (p=0.024). IL-12 caused a non-significant trend towards improvement in airway hyper-responsiveness to histamine, but had no significant effect on the late asthmatic reaction after inhaled allergen challenge. After administration of IL-12, four of 19 patients withdrew prematurely; two with cardiac arrhythmias, one with abnormal liver function, and a single patient with severe flu-like symptoms.

CONCLUSIONS

We have shown that IL-12 lowers numbers of blood and sputum eosinophils, but without any significant effects on airway hyper-responsiveness or the late asthmatic reaction. This questions the role of eosinophils in mediating these reactions, and has important implications for development of new anti-inflammatory treatments.

<em>IL</em>-2-dependent cell lines were established from normal peripheral blood T lymphocytes that express neither CD4 nor CD8 differentiation antigens. CD3+,4-,8- cell lines from 15 different donors failed to react with WT31, an mAb directed against the T cell antigen receptor alpha/beta heterodimer. Anti-Leu-4 mAb was used to isolate the CD3/T cell antigen receptor complex from 125I-labeled CD3+,4-,8- (WT31-) T cells. Using detergent conditions that preserved the CD3/T cell antigen receptor complex, an approximately 90 kD disulfide-linked heterodimer, composed of approximately 45- and approximately 40- (or approximately 37-) kD subunits, was coimmunoprecipitated with the invariant <em>20</em>-29-kD CD3 complex. Analysis of these components by nonequilibrium pH gradient electrophoresis indicated that the approximately 40-kD and approximately 37-kD subunits were similar, and quite distinct from the more basic approximately 45-kD subunit. None of these three subunits reacted with an antibody directed against a beta chain framework epitope. Heteroantiserum against a T cell receptor gamma chain peptide specifically reacted with both the approximately 37- and approximately 40-kD CD3-associated proteins, but not with the approximately 45-kD subunit. CD3+,4-,8- cells failed to transcribe substantial amounts of functional 1.3-kb beta or 1.6-kb alpha mRNA, but produced abundant 1.6-kb gamma mRNA. Southern blot analysis revealed that these CD3+,4-,8- cell lines rearranged both gamma and beta genes, and indicated that the populations were polyclonal. The expression of a CD3-associated disulfide-linked heterodimer on CD3+,4-,8- T cell lines established from normal, adult peripheral blood contrasts with prior reports describing a CD3-associated non-disulfide-linked heterodimer on CD3+/WT31- cell lines established from thymus and peripheral blood obtained from patients with immunodeficiency diseases. We propose that this discrepancy may be explained by preferential usage of the two C gamma genes in T lymphocytes.

Recombinant human tumour necrosis factor alpha (rHuTNF alpha) was shown to inhibit human neutrophil migration in the presence or absence of a chemotactic gradient generated with the tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), at doses of <em>20</em>-100 U/10(6) cells. In contrast, neither recombinant human interleukin-1 alpha (rHu<em>IL</em>-1 alpha), rHu<em>IL</em>-1 beta, human leucocyte-derived <em>IL</em>-1 alpha (1Hu<em>IL</em>-1 alpha) nor 1Hu<em>IL</em>-1 beta contained neutrophil migration inhibition properties. However, both the interleukins (1Hu<em>IL</em>-1 alpha, 1Hu<em>IL</em>-1 beta and rHu<em>IL</em>-1 alpha) and rHuTNF alpha stimulated a neutrophil respiratory burst and significantly elevated the neutrophil respiratory response to fMLP (measured as chemiluminescence and H2O2 production). The stimulatory effects were observed at doses of between 5 and 100 U/5 x 10(5) cells. A characteristic feature of the effects of the cytokines was the range of variation observed in neutrophil responses from different individuals. However, a concentration-related effect was observed with each experiment, delineating suboptimal, optimal and supra-optimal cytokine concentrations. Neutrophils treated with rHuTNF alpha and rHu<em>IL</em>-1 alpha and washed free of exogenous cytokine retained the capacity to show an enhanced response to fMLP. Pretreatment of cells with cytochalasin B enhanced their response to fMLP, and this response was further increased if the cells had also been pretreated with the cytokines. The response to phorbol myristate acetate was also enhanced by rHuTNF alpha and rHu<em>IL</em>-1 alpha. The effects of these cytokines on neutrophils could be abolished by boiling the preparation but not by treating it with polymixin B, suggesting that bacterial lipopolysaccharide was not responsible for the activity of these preparations. The rHu<em>IL</em>-1 alpha increased the release of lysozyme, beta-glucuronidase and myeloperoxidase initiated by cytochalasin B/fMLP, while rHuTNF alpha only increased lysozyme release.

The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [<em>IL</em>]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous <em>IL</em>-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately <em>20</em>% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13-acetate (PMA), anti-CD3, or <em>IL</em>-2. There was a correlation between conjugate size and proliferation with <em>IL</em>-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.