OBJECTIVE

A small number of immune response genes have been consistently associated with the common autoimmune conditions. Recently, a linkage disequilibrium (LD) mapping approach, using tag single nucleotide polymorphisms (SNPs), identified genetic association between type 1 diabetes (T1D) and the interleukin-2 receptor alpha (IL-2Ralpha)/CD25 gene region on chromosome 10p15. Because certain autoimmune diseases, such as autoimmune thyroid disease (AITD) and T1D cluster together in certain families, we sought to determine if the TID-associated CD25 region was also associated with Graves' disease (GD).

METHODS

We performed a case-control association study of 20 tag SNPs.

METHODS

1896 GD patients were collected from seven major centres in the UK and 1822 geographically matched controls from the 1958 British Birth Cohort.

METHODS

The 20 tag SNPs were analysed using a multilocus test to identify an association between GD and the CD25 region. Odds ratios (ORs) were calculated for the tag SNPs, allowing a comparison with previous results for T1D. RESULTS The multilocus test provided statistical evidence of an association between GD and the CD25 region (P = 4.5 x 10(-4)), with the pattern of association of the 20 tag SNPs similar to that found in T1D. CONCLUSIONS Association with GD, as well as that previously reported with T1D, suggests that the CD25 region is acting as a general susceptibility locus for autoimmune disease, and is consistent with a major role for the IL-2-receptor pathway in the development and function of T cells in the control of autoimmunity.

T lymphocytes and immunoregulatory cytokines may be important in the host response to hepatitis C virus (HCV) infection. T-helper type 1 (Th1) cytokines (interleukin [<em>IL</em>]-2, interferon gamma [IFN-gamma]) are required for host antiviral immune responses, including cytotoxic T-cell generation and natural killer cell activation, while T-helper type 2 (Th2) cytokines (<em>IL</em>-4,<em>IL</em>-10) can inhibit the development of these effector mechanisms. In this study, the serum levels of Th1 and Th2 cytokines in patients (n = 23) infected with HCV were measured and compared with biochemical (alanine transaminase [ALT]) and viral (HCV RNA) indicators of infection. Serial cytokine levels were measured in a subset of 11 patients at 1 and 12 weeks during and at 1 week after interferon alfa (IFN-alpha) therapy (n = 33 samples). Levels of circulating <em>IL</em>-2, <em>IL</em>-4, <em>IL</em>-10, and IFN-gamma were significantly elevated in HCV patients versus normal controls (128 vs. 25 pg/mL, 3,045 vs. 29 pg/mL, 2,949 vs. 18 pg/mL, and 307 vs. 24 pg/mL respectively; P < .01). Treatment with IFN-alpha decreased the levels of <em>IL</em>-4 (321 +/- 224 pg/mL), and <em>IL</em>-10 (1,011 +/- 344 pg/mL), which paralleled a decrease in HCV RNA (114 +/- 27 vs. 25 +/- <em>20</em> Eq/ml X 10(5), pre- vs. post-IFN-alpha [12 weeks];P <.05). These findings indicate that an activated T-cell response, as manifest by increased circulating immunoregulatory cytokines, is present in patients with HCV liver disease. Furthermore, treatment with HCV liver disease. Furthermore, treatment with IFN-alpha diminishes the Th2 cytokine response. Thus, modulation of T-cell function and cytokine production may be one mechanism whereby IFN-alpha therapy results in reduced viral burden.

Interleukin 10 (<em>IL</em>-10) is a cytokine with a variety of reported effects including inhibition of monocyte major histocompatibility complex (MHC) class II-dependent antigen presentation, type 1 helper T cell cytokine production, and inhibition of T cell proliferation. Herein we report the effect of <em>IL</em>-10 pretreatment on antigen presentation to tumor- and allo-specific CD8+ cytotoxic T lymphocytes (CTL). Prior incubation of human melanoma cells with recombinant <em>IL</em>-10 (r<em>IL</em>-10) for 48-72 h resulted in a dose-dependent, up to 100% inhibition, of autologous CTL-mediated, HLA-A2.1-restricted, tumor-specific lysis. Allo-specific CTL cytotoxicity against Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) was also inhibited, demonstrating a protective effect also on lymphoid cells. In contrast, <em>IL</em>-10 pretreatment of allogeneic LCL or K562 targets had either no effect or slightly enhanced cytotoxic activity mediated by freshly isolated or <em>IL</em>-2-activated natural killer cells. Flow cytometric analysis with monoclonal antibodies against HLA-A2, or nonpolymorphic determinants of MHC class I proteins, revealed a <em>20</em>-50% reduction in cell-surface expression, whereas intercellular adhesion molecules 1, and 2, and lymphocyte function-associated antigen 3 levels were not affected. In addition, relative to untreated target cells, <em>IL</em>-10 pretreated tumor cells were unaltered in their capacity to affect CTL-mediated lysis by cold target inhibition, demonstrating that the effect of <em>IL</em>-10 is unrelated to the initial binding of CTL to their targets. These results are compatible with an effect of <em>IL</em>-10 on the MHC class I antigen presentation pathway, and suggest a novel mechanism of immune tolerance, based on escape from CTL-mediated tumor and allo-transplant rejection.

Chronic hepatitis C is characterized by a weak or absent hepatitis C virus (HCV)-specific CD4(+) T-cell response in terms of antigen-specific proliferation or interferon gamma (IFN-gamma) secretion. To clarify whether this is due to the absence or functional impairment of antigen-specific CD4(+) T cells we developed an assay that relies on the induced expression of the T-cell activation marker CD25 and is therefore independent from cytokine secretion or proliferation. In 10 of <em>20</em> patients with chronic hepatitis C, a significant number of antigen-specific activated CD4(+) T cells (mean 1.06%/patient; range, 0% to 5.2% of CD4(+) T cells) could be shown, whereas antigen-specific proliferation was present in only 1 of <em>20</em> patients. IFN-gamma secretion was absent in all 13 patients tested. However, significant antigen-specific interleukin 10 (<em>IL</em>-10) and transforming growth factor beta (TGF-beta) secretion was present in 6 of 10 and 3 of 10 patients, respectively. In 8 patients with acute hepatitis C, irrespective of disease outcome, HCV-specific CD4(+) T cells were detected in all patients and at a significantly higher frequency (mean 3.7%/patient; range, 1.16% to 7.17%) in the first weeks of disease. A chronic course of disease was associated either with a loss of both IFN-gamma secretion and proliferation, resembling an anergic state, or a loss of T-cell proliferation followed by a rapid decline in IFN-gamma-producing cells, corresponding to exhaustion of the specific immune response. In conclusion, functional changes of HCV-specific CD4(+) T cells or failure to develop a long-lasting T-helper response may contribute to chronic hepatitis C viral persistence.

Previous studies have shown that airway administration of adenovirus or adenovirus vectors results in a dose-dependent inflammatory response which limits the duration of transgene expression. We explored the possibility that adenovirus infection triggers signal transduction pathways that induce the synthesis of cytokines and thus contribute to the early inflammatory response. Since stimulation of the Raf/mitogen-activated protein kinase (MAPK) pathway activates transcription factors that control the expression of inflammatory cytokines, we examined the activation of this pathway following adenovirus infection. Adenovirus infection induced the rapid activation of Raf-1 and a transient increase in the tyrosine phosphorylation and activation of p42mapk at early times postinfection. Activation of the Raf/MAPK pathway by adenovirus is likely triggered by the infection process, since it occurred rapidly and with various mutant adenoviruses and adenovirus vectors. Moreover, interleukin-8 (<em>IL</em>-8) mRNA accumulation was evident at <em>20</em> min postinfection and was induced even in the presence of cycloheximide. Both MAPK activation and <em>IL</em>-8 production were inhibited by forskolin, a potent inhibitor of Raf-1. These results suggest that adenovirus-induced Raf/MAPK activation contributes to <em>IL</em>-8 production. Adenovirus-induced activation of the Raf/MAPK signaling pathway and <em>IL</em>-8 production may play critical roles in the inflammation observed following in vivo administration of adenovirus vectors for gene therapy.

The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by oxidative stress and pro-inflammatory stimuli and controls the expression of numerous genes involved in the inflammatory response. Dampening NF-kappaB activation and thereby limiting the inflammatory response have been suggested as a potential strategy to prevent chronic inflammatory diseases. In cultured monocytes, anthocyanins isolated from bilberries and black currants (Medox) efficiently suppressed LPS-induced activation of NF-kappaB. Furthermore, we studied the effect of anthocyanin supplementation (Medox, 300 mg/d for 3 wk) in a parallel-designed, placebo-controlled clinical trial (n = 1<em>20</em> men and women aged 40-74 y). Differences were observed in several NF-kappaB related inflammatory mediators in the Medox group compared to placebo. The changes in the NF-kappaB-controlled pro-inflammatory chemokines <em>IL</em>-8, "regulated upon activation, normal T cell expressed and secreted," (RANTES) and IFNalpha (an inducer of NF-kappaB activation) in the Medox group (45, 15, and 40% decreases from baseline, respectively) differed from those in the placebo group (<em>20</em>, 0, and 15% decreases from baseline, respectively) (P < 0.050). Similarly, changes in <em>IL</em>-4 and <em>IL</em>-13, 2 cytokines that mediate pro-inflammatory responses and induce NF-kappaB activation, in the Medox group (60 and 38% decreases from baseline, respectively) tended to differ from those in the placebo group (4 and 6% decreases) (P = 0.056 and, P = 0.089, respectively). These data suggest that anthocyanin supplementation may have a role in the prevention or treatment of chronic inflammatory diseases by inhibition of NF-kappaB transactivation and deceased plasma concentrations of pro-inflammatory chemokines, cytokines, and inflammatory mediators.

Interleukin-1 (<em>IL</em>-1) is a major mediator of inflammation that exerts its biological activities through the <em>IL</em>-1 type I receptor (<em>IL</em>-1RI). The body weights of <em>IL</em>-1RI(-/-) mice of both sexes started to deviate from those of wild-type mice at 5-6 months of age and were <em>20</em>% higher at 9 months of age. Visceral and subcutaneous fat mass, measured by dual-energy X-ray absorptiometry and magnetic resonance imaging, was markedly (1.5- to 2.5-fold) increased. Lean body mass and crown-rump length were also slightly (11 and 5%, respectively) increased, as was serum IGF-I. Obese <em>IL</em>-1RI(-/-) mice were insulin resistant, as evidenced by hyperinsulinemia, decreased glucose tolerance, and insulin sensitivity. To elucidate the mechanisms for the development of obesity, young pre-obese <em>IL</em>-1RI(-/-) mice were investigated. They showed decreased suppression of body weight and food intake in response to systemic leptin treatment. The decreased leptin responsiveness was even more pronounced in older obese animals. Moreover, spontaneous locomotor activity and fat utilization, as measured by respiratory quotient, were decreased in pre-obese <em>IL</em>-1RI(-/-) mice. In conclusion, lack of <em>IL</em>-1RI-mediated biological activity causes mature-onset obesity. This obese phenotype is preceded by decreased leptin sensitivity, fat utilization, and locomotor activity.

Cytokines may play an important role in the pathophysiology of traumatic brain injury (TBI) in children. Interleukin-6 (<em>IL</em>-6) is a proinflammatory cyotkine that plays a role in regenerative processes within the central nervous system (CNS), whereas interleukin-10 (<em>IL</em>-10) is an antiinflammatory cytokine. Both have been measured in serum and cerebrospinal fluid (CSF) as an index of the degree of inflammation in diseases, including sepsis and meningitis. We hypothesized that both <em>IL</em>-6 and <em>IL</em>-10 would be increased in the CSF of children after severe TBI. Fifteen children who sustained severe TBI (Glascow Coma Score [GCS] < or = 7) were studied. Standard neurointensive care was provided. Ventricular CSF collected the first 3 days after TBI was analyzed for <em>IL</em>-6 and <em>IL</em>-10 concentrations by ELISA. Controls were <em>20</em> children who were evaluated for meningitis with diagnostic lumbar puncture subsequently found to have no CSF pleocytosis and negative cultures. <em>IL</em>-6 was increased in children after TBI versus controls on all days studied (day 1, 3158.2 +/- 621.8 pg/ml; day 2, 1111.6 +/- 337.0 pg/ml; day 3, 826.7 +/- 193.5 pg/ml vs. <em>20</em>.6 +/- 5.8 pg/ml, p < 0.0001, Mann-Whitney Rank Sum). <em>IL</em>-10 was increased in children after TBI vs controls on all days studied (day 1, 47.2 +/- 12.9 pg/ml; day 2, 21.0 +/- 6.7 pg/ml; day 3, 15.5 +/- 5.9 pg/ml vs. 8.9 +/- 7.5 pg/ml, p < 0.01). Increased <em>IL</em>-10 concentrations were independently associated with age < 4 years and mortality (p = 0.004 and 0.04, respectively, multivariate linear model). This study demonstrates that <em>IL</em>-6 is increased after TBI in children to levels similar to those reported in adults and is the first to show that <em>IL</em>-10 is increased in CSF of humans after TBI. These data suggest that there may be an age-dependent production of <em>IL</em>-10 after TBI in children.

Isolating rats immediately after conditioning impairs contextual but not auditory-cue fear conditioning. The reported experiments examine the involvement of brain interleukin-1beta (<em>IL</em>-1beta) in the impairment in contextual fear conditioning caused by social isolation. As measured by the conditioned freezing response, 5 h of social isolation after conditioning, impaired contextual but not auditory-cue fear conditioning in adult male Sprague-Dawley rats. Social isolation for 1 or 3 h after conditioning also increased <em>IL</em>-1beta protein in the hippocampus and cerebral cortex. No differences in <em>IL</em>-1beta protein levels were found in the pituitary or the hypothalamus. Intracerebroventricular (ICV) <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra) given after conditioning prevented the impairment in contextual fear conditioning caused by isolation. ICV <em>IL</em>-1ra had no effect on auditory-cue fear conditioning in these same animals, nor did it affect the level of contextual fear conditioning displayed by home cage controls. Like isolation, ICV <em>IL</em>-1beta (10 or <em>20</em> ng) after conditioning also impaired contextual but not auditory-cue fear conditioning. These results suggest that increased levels of brain <em>IL</em>-1beta play a role in producing the impairment in contextual fear conditioning produced by social isolation. These findings also add to the generality of the idea that stressors induce <em>IL</em>-1beta activity in the brain and that <em>IL</em>-1beta may play physiological roles in the uninjured brain.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, <em>20</em>% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, <em>IL</em>-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

BACKGROUND

Mimetics of bacterial DNA, given orally or subcutaneously, protect mice from experimental colitis via a toll-like receptor (TLR)-9-dependent mechanism. The goal of the study was to define whether synthetic viral RNA, polyinosinic acid:cytidylic acid [poly(I:C)], which is also a potent immunomodulator, might also affect murine colitis and, if so, define whether such effects were mediated by TLR3, which is one of at least 4 known receptors for this viral RNA analog.

METHODS

Mice (C57BL6, <em>IL</em>-10KO, or TLR3 KO) were administered 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days. Two hours before treatment with DSS, mice were given phosphate-buffered saline (PBS) or poly(I:C) <em>20</em> mug subcutaneously (s.c.), or 100 mug intragastrically (i.g.).

RESULTS

In wildtype mice s.c. administration of poly(I:C) dramatically protected against DSS-induced colitis as assessed by every parameter analyzed, which included body weight, rectal bleeding, colonic myeloperoxidase, histopathology, serum keratinocyte-derived chemokine, serum amyloid A, and lipocalin-2. In contrast, i.g. administration of poly(I:C) offered no protection in this colitis model nor did its administration activate the innate immune system as assessed by serologic parameters. Subcutaneous poly(I:C) protected against DSS-induced colitis equally well in C57BL6 and IL-10KO mice, indicating that this antiinflammatory cytokine is not required for such protection. Protection against colitis given by poly(I:C) treatment was ablated in TLR3 KO, indicating that the protective action of this viral RNA analog was mediated by this receptor.

CONCLUSIONS

Activation of TLR3 on cells that are accessible by systemic, but not oral, administration of synthetic viral RNA results in protection against the acute inflammation that can ensue upon damage of the gut epithelium. Thus, this viral RNA analog, which is under clinical trials for other inflammatory disorders (e.g., lupus), may also have therapeutic value for inflammatory bowel disease.

<em>IL</em>-15 uses the heterotrimeric receptor <em>IL</em>-2/<em>IL</em>-15Rβ and the γ chain shared with <em>IL</em>-2 and the cytokine-specific <em>IL</em>-15Rα. Although <em>IL</em>-15 shares actions with <em>IL</em>-2 that include activation of natural killer (NK) and CD8 T cells, <em>IL</em>-15 is not associated with capillary leak syndrome, activation-induced cell death, or with a major effect on the number of functional regulatory T cells. To prepare for human trials to determine whether <em>IL</em>-15 is superior to <em>IL</em>-2 in cancer therapy, recombinant human <em>IL</em>-15 (rh<em>IL</em>-15) was produced under current good manufacturing practices. A safety study in rhesus macaques was performed in 4 groups of 6 animals each that received vehicle diluent control or rh<em>IL</em>-15 at 10, <em>20</em>, or 50 μg/kg/d IV for 12 days. The major toxicity was grade 3/4 transient neutropenia. Bone marrow examinations demonstrated increased marrow cellularity, including cells of the neutrophil series. Furthermore, neutrophils were observed in sinusoids of enlarged livers and spleens, suggesting that <em>IL</em>-15 mediated neutrophil redistribution from the circulation to tissues. The observation that <em>IL</em>-15 administration was associated with increased numbers of circulating NK and CD8 central and effector-memory T cells, in conjunction with efficacy studies in murine tumor models, supports the use of multiple daily infusions of rh<em>IL</em>-15 in patients with metastatic malignancies.

The cytokine interleukin-1 (<em>IL</em>-1) has been implicated in ischaemic, excitotoxic and traumatic brain damage in rodents. The naturally occurring <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra) markedly reduces neuronal injury in these conditions. However, the effects of <em>IL</em>-1ra on focal, transient cerebral ischaemia in the rat, which is of major clinical relevance, have not been reported. The objectives of this study were to test the effects of <em>IL</em>-1ra on cell death after temporary cerebral ischaemia, and to investigate the therapeutic time window for <em>IL</em>-1ra treatment. Ischaemia was induced by temporary (60 min) occlusion of the middle cerebral artery (MCAO) in rats, via surgical insertion (and subsequent removal) of a thread into the internal carotid artery. Damage was quantified at various times after MCAO to investigate the temporal progression of damage and establish an appropriate time to assess the effects of <em>IL</em>-1ra on cell death. Cell death was complete 18-24 h after temporary MCAO. Intracerebroventricular injection of <em>IL</em>-1ra (10 microg) at the time of MCAO and 60 min later reduced the lesion volume measured 24 h (57% reduction) or 48 h (52% reduction) after MCAO. Cell death was also significantly reduced when <em>IL</em>-1ra (<em>20</em> microg) was administered as a single injection, 1 h (47%), 2 h (57%) or 3 h (46%) after MCAO, when compared to vehicle. These data show that <em>IL</em>-1ra markedly reduces cell death even when administration is delayed until 3 h after induction of reversible, focal cerebral ischaemia in the rat, and support our proposal that <em>IL</em>-1ra may be of therapeutic benefit in stroke.

<em>IL</em>-10 is an immunosuppressive cytokine in the immune system. It was in clinical trial as an anti-inflammatory therapy for inflammatory bowel disease and various autoimmune diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. <em>IL</em>-19 belongs to the <em>IL</em>-10 family, which includes <em>IL</em>-10, <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, melanoma differentiation-associated gene (MDA-7, <em>IL</em>-24), and AK155 (<em>IL</em>-26). Despite a partial homology in their amino acid sequences, they are dissimilar in their biologic functions. Little is known about the biologic function and gene regulation of <em>IL</em>-19. To understand the gene regulation of human <em>IL</em>-19, we identified a human <em>IL</em>-19 genomic clone and analyzed its promoter region. Five fusion genes containing different regions upstream of exon 1 linked to a luciferase reporter gene were expressed in the canine kidney epithelial-like Madin-Darby canine kidney cells. A fusion gene containing 394 bp showed luciferase activity 7- to 8-fold higher than the negative control of the promoterless fusion gene. We also isolated a full-length mouse cDNA clone. Mouse <em>IL</em>-19 shared 71% amino acid identity with human <em>IL</em>-19. Treatment of monocytes with mouse <em>IL</em>-19 induced the production of <em>IL</em>-6 and TNF-alpha. It also induced mouse monocyte apoptosis and the production of reactive oxygen species. Taken together, our results indicate that mouse <em>IL</em>-19 may play some important roles in inflammatory responses because it up-regulates <em>IL</em>-6 and TNF-alpha and induces apoptosis.

Proinflammatory Th17 cells and CD4(+)CD25(+) regulatory T (Treg) cells are two newly identified T lymphocyte subsets, which have opposite effects on autoimmunity and inflammation. To assess the Th17/Treg pattern and cytokine microenvironment in peripheral blood of patients with RA, we included 66 RA patients and <em>20</em> healthy volunteers. Of all these subjects, peripheral Th17 and Treg frequencies were analyzed by flow cytometry (FCM) and the plasma levels of interleukin (<em>IL</em>)-17, 23, 6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1 were detected by ELISA. The results demonstrated that RA patients revealed an obvious increase in peripheral Th17 frequencies and levels of Th17-related cytokines (<em>IL</em>-17, <em>IL</em>-23, <em>IL</em>-6, TNF-α) while a significant decrease in Treg frequencies and Treg-related cytokine (TGF-β1) levels when compared with healthy people. Our study indicated that development of RA is associated with peripheral Th17/Treg imbalance and characterized by a proinflammatory cytokine microenvironment, which supports continuing generation of Th17 cells.

This paper demonstrates how the shape and size of gold nanoparticles (AuNPs) affect immunological responses in vivo and in vitro for the production of antibodies for West Nile virus (WNV). We prepared spherical (<em>20</em> and 40 nm in diameter), rod (40 × 10 nm), and cubic (40 × 40 × 40 nm) AuNPs as adjuvants and coated them with WNV envelope (E) protein. We measured anti-WNVE antibodies after inoculation of these WNVE-coated AuNPs (AuNP-Es) into mice. The 40 nm spherical AuNP-Es (Sphere40-Es) induced the highest level of WNVE-specific antibodies, while rod AuNP-Es (Rod-Es) induced only 50% of that of Sphere40-E. To examine the mechanisms of the shape-dependent WNVE antibody production, we next measured the efficiency of cellular uptake of AuNP-Es into RAW264.7 macrophage cells and bone-marrow-derived dendritic cells (BMDCs) and the subsequent cytokine secretion from BMDCs. The uptake of Rod-Es into the cells proceeded more efficiently than those of Sphere-Es or cubic WNVE-coated AuNPs (Cube-Es), suggesting that antibody production was not dependent on the uptake efficiency of the different AuNP-Es. Cytokine production from BMDCs treated with the AuNP-Es revealed that only Rod-E-treated cells produced significant levels of interleukin-1β (<em>IL</em>-1β) and interleukin-18 (<em>IL</em>-18), indicating that Rod-Es activated inflammasome-dependent cytokine secretion. Meanwhile, Sphere40-Es and Cube-Es both significantly induced inflammatory cytokine production, including tumor necrosis factor-α (TNF-α), <em>IL</em>-6, <em>IL</em>-12, and granulocyte macrophage colony-stimulating factor (GM-CSF). These results suggested that AuNPs are effective vaccine adjuvants and enhance the immune response via different cytokine pathways depending on their sizes and shapes.

Interleukin (<em>IL</em>)-2 is used in the immunotherapy of patients with certain cancer and HIV infection. <em>IL</em>-2 treatment reliably results in 16% to <em>20</em>% objective clinical response rate in cancer patients, with significant durability of responses in selected patients. However, the mechanisms of therapeutic activity in responding versus nonresponding patients remain poorly understood. CD4(+)CD25(+)FOXP3(+) regulatory T (Treg) cells contribute to immunosuppressive networks in human tumors. We treated 31 ovarian cancer patients with <em>IL</em>-2. We show that administration of <em>IL</em>-2 induces the proliferation of existent Treg cells in patients with ovarian cancer. The potency of Treg cell proliferation is negatively determined by the initial prevalence of Treg cells, suggesting that Treg cells are a factor for self-controlling Treg cell proliferation. After <em>IL</em>-2 cessation, the number of Treg cells more efficiently dropped in clinical responders than nonresponders. Furthermore, <em>IL</em>-2 treatment stimulates chemokine receptor CXCR4 expression on Treg cells, enables Treg cell migration toward chemokine CXCL12 in the tumor microenvironment, and may enforce Treg cell tumor accumulation. Our findings support the concept that administration of <em>IL</em>-2 numerically and functionally affects the Treg cell compartment. These data provide an important insight in evaluating the clinical benefit and therapeutic prediction of <em>IL</em>-2 treatment in patients with cancer.

Mammals express ∼<em>20</em> different connexins, the main gap junction forming proteins in mammals, and 3 pannexins, homologs of innexins, the main gap junction forming proteins in invertebrates. In both classes of gap junction, each channel is formed by two hemichannels, one contributed by each of the coupled cells. There is now general, if not universal, agreement that hemichannels of both classes can open in response to various physiological and pathological stimuli when they are not apposed to another hemichannels and face the external milieu. Connexin (and likely pannexin) hemichannel permeability is consistent with that of the cell-cell channels and open hemichannels can be a release site for relatively large molecules such as ATP and glutamate, which can serve as transmitters between cells. Here we describe three experimental paradigms in which connexin and pannexin hemichannel signaling occurs. (1) In cultures of spinal astrocytes FGF-1 causes the release of ATP, and ATP causes opening of pannexin hemichannels, which then release further ATP. Subsequently, several hours later, connexin hemichannels are also opened by an unknown mechanism. Release of ATP appears to become self sustaining through action of P2X7 receptors to open pannexin hemichannels and then connexin hemichannels, both of which are ATP permeable. (2) Spinal cord injury by dropping a small weight on the exposed cord is followed by release of ATP in the region surrounding the primary lesion. This release is greatly reduced in a mouse in which Cx43 is knocked down in the astrocytes. Application of FGF-1 causes a similar release of ATP in the uninjured spinal cord, and an inhibitor of the FGF-1 receptor, PD173074, inhibits both FGF-1 and injury-induced release. Reduction in ATP release is associated with reduced inflammation and less secondary expansion of the lesion. (3) Cortical astrocytes in culture are permeabilized by hypoxia, and this effect is increased by high or zero glucose. The mechanism of permeabilization is opening of Cx43 hemichannels, which can lead to cell death. Activated microglia secrete TNF-α and <em>IL</em>-1β, which open connexin hemichannels in astrocytes. Astrocytes release ATP and glutamate which can kill neurons in co-culture through activation of neuronal pannexin hemichannels. These studies implicate two kinds of gap junction hemichannel in inflammatory responses and cell death. This article is part of a Special Issue entitled Electrical Synapses.

Most attacks of acute pancreatitis are mild and self-limiting. In 10-<em>20</em>% of the cases, however, severe disease with multiple systemic complications develops. During the last few years it has been recognized that activated leukocytes have an important role in the multisystem involvement during acute pancreatitis. Activated leukocytes are thus a pathogenetic factor in the severity of the disease. Activation of polymorphonuclear granulocytes (PMNs) and of monocytes/macrophages is an early event during severe acute pancreatitis. Factors released by activated leukocytes therefore reflect the severity of the disease. Three independent studies have shown that released PMN-elastase is a reliable early prognostic marker that permits correct classification of 80-95% of the patients within the first 24-48 hours. Interleukin-6 (<em>IL</em>-6), mainly secreted by activated monocytes/macrophages, is also an early prognostic parameter (shown in one study), but is not superior to PMN-elastase. Leukocyte activation markers are more reliable than multiple scoring systems in the assessment of the severity of acute pancreatitis. Compared with PMN-elastase or <em>IL</em>-6, increased plasma concentrations of such acute-phase proteins as alpha-1-antitrypsin or CRP, and consumption of the protease inhibitor alpha-2-macroglobulin, are later events that can be detected only 1 to 4 days later. Comparison of the various inflammatory parameters suggests that PMN-elastase is the best early and reliable prognostic marker in acute pancreatitis. The reviewed data underscore the role of activated leukocytes in the pathogenesis of complicated acute pancreatitis.

OBJECTIVE

The anti-inflammatory limb of the immune response is crucial for dampening inflammation. Spontaneous parturition at term and preterm labor (PTL) are mediated by inflammation in the cervix, membranes, and myometrium. This study focuses on the changes in the amniotic fluid concentrations of the anti-inflammatory cytokine interleukin (IL)- 10. The objectives of this study were to determine whether there is a relationship between amniotic fluid concentrations of IL-10 and gestational age, parturition (at term and preterm), and intra-amniotic infection/inflammation (IAI).

METHODS

A cross-sectional study was conducted including 301 pregnant women in the following groups: (1) mid-trimester of pregnancy who delivered at term (n = 112); (2) mid-trimester who delivered preterm neonates (n = 30); (3) term not in labor without IAI (n = 40); (4) term in labor without IAI (n = 24); (5) term in labor with IAI (n = 20); (6) PTL without IAI who delivered at term (n = 31); (7) PTL without IAI who delivered preterm (n = 30); (8) PTL with IAI who delivered preterm (n = 14). IL-10 concentrations in amniotic fluid were determined by a specific and sensitive immunoassay. Non-parametric statistics were used for analysis.

RESULTS

(1) IL-10 was detectable in amniotic fluid and its median concentration did not change with gestational age from mid-trimester to term. (2) Patients in labor at term had a significantly higher median amniotic fluid IL-10 concentration than that of patients at term not in labor (p = 0.04). (3) Women at term in labor with IAI had a significantly higher median amniotic fluid IL-10 concentration than that of patients at term in labor without IAI (p = 0.02). (4) Women with PTL and IAI who delivered preterm had a significantly higher median amniotic fluid concentration of IL-10 than those without IAI who delivered preterm and than those who delivered at term (p = 0.009 and p < 0.001, respectively). (5) Among patients with preterm labor without IAI, those who delivered preterm had a significantly higher median amniotic fluid IL-10 concentration than those who delivered at term (p = 0.03).

CONCLUSIONS

The anti-inflammatory cytokine IL-10 is detectable in the amniotic fluid of normal pregnant women. Spontaneous parturition at term and in preterm gestation is associated with increased amniotic fluid concentrations of IL-10. IAI (preterm and at term) is also associated with increased amniotic fluid concentrations of IL-10. We propose that IL-10 has a role in the regulation of the immune response in vivo by initiating actions that dampen inflammation.

A Shwartzman-like reaction was elicited in rabbits by preparing the skin with intradermal injections of recombinant human tumor necrosis factor alpha (TNF alpha) and recombinant human interleukin-1 (<em>IL</em>-1 alpha or beta). The animals were challenged intravenously with endotoxin or by intravascular activation of complement with immune complexes or zymosan 18 hours later and were sacrificed after another 2 hours. Animals challenged with saline did not develop Shwartzman-like reactions. The sites prepared with endotoxin or with either form of <em>IL</em>-1 plus TNF alpha developed visible hemorrhage, whereas sites injected with either <em>IL</em>-1 or TNF alpha alone did not. Hemorrhage and microthrombosis were quantitated with 59Fe-labeled erythrocytes and 111In-platelets for 2 hours after the intravenous challenge, and the findings confirmed the observations made on gross inspection. Dermal sites prepared with the cytokines and challenged intravenously with endotoxin, immune complexes, or zymosan exhibited some diffuse hemorrhage and an intense erythrocyte extravasation around distended vessels, along skin appendages, and the panniculus carnosus muscle. The lumens of many large and postcapillary venules contained aggregates of platelets and leukocytes. These changes were superimposed on those seen at prepared sites (leukocyte infiltration). By electron microscopy fibrin was demonstrable in association with the formed elements of the blood. Histologic examination of the 18-hour-old preparative lesions or <em>20</em>-hour-old lesions of saline-"challenged" animals revealed accumulation of leukocytes in the dermis, predominantly neutrophils. This accumulation was sparse at sites treated with only <em>IL</em>-1 or TNF alpha, but very intense at sites treated with both <em>IL</em>-1 and TNF alpha or with endotoxin. These observations were confirmed quantitatively by measuring the accumulation of 51Cr-labeled neutrophils for 2 hours after injection. The potency of <em>IL</em>-1 alpha was comparable to that in our earlier report, and TNF alpha was about three log times less potent. Sites treated with both <em>IL</em>-1 alpha and TNF alpha resulted in 69% greater neutrophil emigration than the additive response elicited by each cytokine. The reported findings implicate a synergism between <em>IL</em>-1 and TNF alpha in the induction of both the inflammatory reaction (preceding the Shwartzman reaction) and the thrombohemorrhagic component of the Shwartzman reaction proper.

BACKGROUND

Pre-gravid obesity is associated with increased morbidity and mortality for both mother and offspring. Recent studies have demonstrated a heightened inflammatory response both systemically and locally within the adipose and placental tissue in women with pre-gravid obesity, which may play a role in mediating the adverse pregnancy outcomes. The aim of this study was to characterise the maternal and placental inflammatory status and investigate associated changes in placental structure in obese women.

METHODS

The pro-inflammatory status of a cohort of 47 non-obese (BMI <em>20</em>-25 kg/m(2)) and 33 obese (≥30 kg/m(2)) women was characterised by measuring maternal circulating levels and placental gene expression of pro-inflammatory cytokines, and quantifying immune cell populations within the placenta. The effect of pre-gravid obesity on placental structure was investigated by examining placental maturity, vessel density, the formation of syncytial knots and sprouts, and the degree of fibrin deposition, chorangiosis and muscularisation of vessel walls.

RESULTS

Maternal obesity was associated with significantly greater IL-1β (p < 0.05), IL-8 (p < 0.05), MCP-1 (p < 0.001) and CXCR2 (p < 0.05) mRNA expression within the placenta and higher circulating maternal levels of IL-6 (3.30 ± 0.38 vs. 1.77 ± 0.15 pg/ml) (p < 0.001) compared with non-obese women. There were no differences in the number of CD14(+), CD68(+) cells or neutrophils within the placental villi of non-obese and obese women. However there were significantly higher numbers of neutrophils within the interstitial space (p < 0.05). Greater muscularity of placental vessel walls was associated with maternal obesity (p = 0.03), however no other associated structural changes were observed.

CONCLUSIONS

Our findings show that although pre-gravid obesity was associated with greater expression of placental pro-inflammatory cytokines and higher circulating IL-6 in pregnancy, there were no major differences in immune cell populations within the placental villi and only a greater degree of muscularity in the vessel walls.

OBJECTIVE

Serum testosterone levels are known to inversely correlate with insulin sensitivity and obesity in men. Furthermore, there is evidence to suggest that testosterone replacement therapy reduces insulin resistance and visceral adiposity in type 2 diabetic men. Adipocytokines are hormones secreted by adipose tissue and contribute to insulin resistance. We examined the effects of testosterone replacement treatment on various adipocytokines and C-reactive protein (CRP) in type 2 diabetic men.

METHODS

Double-blinded placebo-controlled crossover study in <em>20</em> hypogonadal type 2 diabetic men. Patients were treated with testosterone (sustanon <em>20</em>0 mg) or placebo intramuscularly every 2 weeks for 3 months in random order followed by a washout period of 1 month before the alternate treatment phase.

METHODS

Leptin, adiponectin, resistin, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and CRP levels were measured before and after each treatment phase. Body mass index (BMI) and waist circumference were also recorded.

RESULTS

At baseline, leptin levels significantly correlated with BMI and waist circumference. There was a significant inverse correlation between baseline IL-6 and total testosterone (r=-0.68; P=0.002) and bioavailable testosterone levels (r=-0.73; P=0.007). CRP levels also correlated significantly with total testosterone levels (r=-0.59; P=0.01). Testosterone treatment reduced leptin (-7141.9 +/- 1461.8 pg/ml; P=0.0001) and adiponectin levels (-<em>20</em>75.8 +/- 852.3 ng/ml; P=0.02). There was a significant reduction in waist circumference. No significant effects of testosterone therapy on resistin, TNF-alpha, IL-6 or CRP levels were observed.

CONCLUSIONS

Testosterone replacement treatment decreases leptin and adiponectin levels in type 2 diabetic men. Moreover, low levels of testosterone in men are associated with pro-inflammatory profile, though testosterone treatment over 3 months had no effect on inflammatory markers.

Microglial cells are the immune-competent elements of the brain. They not only express receptors for chemokines and cytokines but also for neurotransmitters such as GABA [Charles et al., Mol. Cell Neurosci. 24 (<em>20</em>03) 214], glutamate [Noda et al., J. Neurosci. <em>20</em> (<em>20</em>00) 251], and adrenaline [Mori et al., Neuropharmacology 43 (<em>20</em>02) 1026]. Here we report the functional expression of dopamine receptors in mouse and rat microglia, in culture and brain slices. Using the patch clamp technique as the functional assay we identified D1- and D2-like dopamine receptors using subtype-specific ligands. They triggered the inhibition of the constitutive potassium inward rectifier and activated potassium outward currents in a subpopulation of microglia. Chronic dopamine receptor stimulation enhanced migratory activity and attenuated the lipopolysaccharide (LPS)-induced nitric oxide (NO) release similar as by stimulation of adrenergic receptors. While, however, noradrenaline attenuated the LPS-induced release of TNF-alpha and <em>IL</em>-6, dopamine was ineffective in modulating this response. We conclude that microglia express dopamine receptors which are distinct in function from adrenergic receptors.