Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of <em>IL</em>-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5'-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human <em>IL</em>-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-d -ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of <em>IL</em>-10. The differential effect of these drugs on monocyte <em>IL</em>-12 and <em>IL</em>-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue's metabolites. Moreover, CGS-21680 inhibited <em>IL</em>-12 production independently of endogenous <em>IL</em>-10 induction, because anti-<em>IL</em>-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on <em>IL</em>-12 production. The phosphodiesterase inhibitor Ro <em>20</em>-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits <em>IL</em>-12 and stimulates <em>IL</em>-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.

The mda-7 gene (approved gene symbol IL-fold) of serum IL-6, IL-10, and TNF-alpha were observed. Significantly higher elevations of IL-6 and TNF-alpha were observed in patients who responded clinically to INGN 241. Patients also showed marked increases of CD3+CD8+ T cells posttreatment, suggesting that INGN 241 increased systemic TH1 cytokine production and mobilized CD8+ T cells. Intratumoral delivery of INGN 241 induced apoptosis in a large volume of tumor and elicited tumor-regulatory and immune-activating events that are consistent with the preclinical features of MDA-7/IL-24.

Previous studies compared uptake by dendritic cells (DC) of <em>20</em>, 40, 100, <em>20</em>0, 500, 1000, and <em>20</em>00 nm beads in vivo. When beads were used as antigen carriers, bead size influenced antibody responses and induction of IFN-gamma-producing CD4 and CD8 T cells. Beads of 40-50 nm were taken up preferentially by DC and induced particularly strong immunity. Herein, we examine immunity induced by minute differences in nanobead size, specifically within a narrow viral-sized range (<em>20</em>, 40, 49, 67, 93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells as demonstrated by the production of IFN-gamma or <em>IL</em>-4. In vivo uptake by DC was assessed for selected sizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptide epitope (SIINFEKL) were tested. After one immunization with beads-OVA, IFN-gamma responses to both OVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, in contrast, <em>IL</em>-4 responses to OVA were higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm). Thus IFN-gamma induction from CD8 T cells was limited to 40-49 nm beads, while CD4 T cell activation and <em>IL</em>-4 were induced by 93-123 nm beads-OVA. After two immunizations, there were comparable high levels of IFN-gamma produced with 40 and 49 beads and <em>IL</em>-4 reactivity was still higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range of bead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus (RSV) depends on strong IFN responses, while <em>IL</em>-4 responses are reported to cause asthma-like symptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for a suitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1 (internal capsid), one immunization with G88 induced high levels of IFN-gamma and low levels of <em>IL</em>-4. <em>IL</em>-4 increased with two immunizations. Beads-M2.1 induced only moderate levels of IFN-gamma and low titer antibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads and challenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered from weight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive control mice. These results show that precise selection of nanobead size for vaccination can influence the type 1/type 2 cytokine balance after one immunization, and this will be useful in the development of effective vaccines against common human pathogens such as RSV.

OBJECTIVE

Both obesity and the metabolic syndrome (MetS) have been independently linked with increased oxidative and inflammatory stress. This study tested the hypothesis that obesity with MetS is associated with greater oxidative and inflammatory burden compared with obesity alone.

METHODS

Forty-eight normal-weight and 40 obese (<em>20</em> without MetS; <em>20</em> with MetS) adults were studied. MetS was defined according to National Cholesterol Education Program Adult Treatment Panel III criteria. Plasma concentrations of oxidized low-density lipoprotein, C-reactive protein, tumor necrosis factor-alpha, interleukin (<em>IL</em>)-6, and <em>IL</em>-18 were determined by enzyme immunoassay.

RESULTS

Plasma biomarkers of oxidative stress and inflammation were lowest in normal-weight controls. Of note, obese MetS adults demonstrated significantly higher plasma concentrations of oxidized low-density lipoprotein (62.3 +/- 3.2 vs. 54.0 +/- 4.0 U/L; p < 0.05), C-reactive protein (3.0 +/- 0.6 vs. 1.5 +/- 0.3 mg/L; p < 0.01), tumor necrosis factor-alpha (2.1 +/- 0.1 vs. 1.6 +/- 0.1 pg/mL; p < 0.05), IL-6 (2.8 +/- 0.4 vs. 1.4 +/- 0.2 pg/mL; p < 0.01), and IL-18 (253 +/- 16 vs. 199 +/- 16 pg/mL; p < 0.01), compared with obese adults without MetS.

CONCLUSIONS

These results suggest that MetS heightens oxidative stress and inflammatory burden in obese adults. Increased oxidative and inflammatory stress may contribute to the greater risk of coronary heart disease and cerebrovascular disease in obese adults with MetS.

Serum samples drawn at diagnosis from 174 myeloma patients were analyzed for the presence of the heparan [corrected] sulfate proteoglycan, syndecan-1. Syndecan-1 was elevated in 79% of patients (median, 643 units/mL) compared with 40 healthy controls (median, 128 units/mL), P <.0001. Serum syndecan-1 correlated with the following: serum creatinine, secretion of urine M-component over the course of 24 hours, soluble interleukin-6 (<em>IL</em>-6) receptor, C-terminal telopeptide of type I collagen, beta(2)-microglobulin, percentage of plasma cells in the bone marrow, disease stage, and serum M-component concentration. In order to evaluate syndecan-1 as a prognostic marker in multiple myeloma, it was entered into a multivariate Cox regression model. Data from 138 patients were available for this analysis. As a continuous variable, syndecan-1 was an independent prognostic parameter in addition to serum beta(2)-microglobulin and World Health Organization performance status. When syndecan-1 was dichotomized by the best cutoff (66th percentile, 1170 units/mL), the survival difference between the groups was highly significant: "high" syndecan-1 group had a median survival of <em>20</em> months, and the "low" syndecan-1 group had a median of 44 months (P <.0001). We conclude that syndecan-1 is a new independent prognostic parameter in multiple myeloma, and its role in prognostic classification systems should be further investigated. (Blood. <em>20</em>00;95:388-392)

OBJECTIVE

Imbalances in the genetically controlled pro- and anti-inflammatory cytokine production may promote ongoing low-grade inflammation after an acute gastroenteritis, and subsequently, irritable bowel syndrome (IBS) (post-infectious IBS, PI-IBS). We studied gene promoter single nucleotide polymorphisms (SNPs) of tumor necrosis factor-alpha (TNF-alpha, pro-inflammatory) and interleukin-10 (IL-10, anti-inflammatory) in IBS patients and controls.

METHODS

DNA was extracted from peripheral blood leucocytes of 111 IBS patients and 162 healthy controls. Genotype and allele frequencies were assessed by analyzing SNPs at position -308 (TNF-alpha) and -1082 and -819 (IL-10).

RESULTS

Homozygous high producers for TNF-alpha (A/A) were rare (overall prevalence 2.6%). The heterozygous TNF-alpha genotype (G/A, high producer) was significantly more prevalent in IBS compared to controls (41%vs 26%, p= 0.02). More patients (41%) than controls (30%) were positive for the A allele (p= 0.044; odds ratio (OR) 1.68, 95% confidence interval (CI) 1.01-2.79), with a similar trend for diarrhea (54%) versus constipation and alternating subtypes (<33%, p= 0.079), but not for subgroups according to a history of acute gastroenteritis. IL-10 genotypes were similarly distributed in patients and controls for both SNPs. Possession of a high producer TNF-alpha and a low producer IL-10 genotype were significantly more prevalent in IBS (9%) versus controls (3%, p= 0.035; OR 3.11, 95% CI 1.03-9.36) and in diarrhea (20%) compared to other IBS subtypes (<4%, p= 0.026).

CONCLUSIONS

Our results support the emerging hypothesis that genetically determined immune activity plays a role in the pathophysiology of IBS. Future studies in larger, clinically relevant, IBS subgroups are warranted to establish definite associations with cytokine gene polymorphisms.

Despite major progress in the treatment of rheumatoid arthritis (RA), strong unmet medical need remains, as only a minor proportion of patients reach sustained clinical remission. New approaches are therefore necessary, and include manipulation of regulatory T cells, which might be able to restore the disturbed immune system and could even lead to a cure if this restored regulation were to prove sustainable. Logistical and conceptual problems, however, beset this attractive therapeutic approach, including difficulties with ex vivo expansion of cells, specificity of targeting and the optimal time point of administration. Therefore, alternative avenues are being investigated, such as targeting B-cell effector functions and newly identified proinflammatory cytokines. On the basis of success with B-cell depleting therapy using anti-CD<em>20</em> agents, further treatment modalities are now exploring direct or indirect interference in B-cell-mediated immunity with the use of agents directed against other B-cell surface molecules. Novel approaches target intracellular B-cell signalling and regulatory B cells. New cytokine-directed therapies target important proinflammatory mediators such as GM-CSF, new members of the <em>IL</em>-1 family, <em>IL</em>-6 and its receptor, <em>IL</em>-17, <em>IL</em>-<em>20</em>, <em>IL</em>-21, <em>IL</em>-23 as well as synovium-specific targets. This article reviews these emerging cell and cytokine targets with special focus on biologic agents, some of which might reach the clinic soon whereas others will require considerable time in development. Nevertheless, these exciting new approaches will considerably enhance our repertoire in the battle against this potentially devastating disease.

Research over the last decade has revealed that CYP11A1 can hydroxylate the side chain of vitamin D3 at carbons 17, <em>20</em>, 22 and 23 to produce at least 10 metabolites, with <em>20</em>(OH)D3, <em>20</em>,23(OH)2D3, <em>20</em>,22(OH)2D3, 17,<em>20</em>(OH)2D3 and 17,<em>20</em>,23(OH)3D3 being the main products. However, CYP11A1 does not act on 25(OH)D3. The placenta, adrenal glands and epidermal keratinocytes have been shown to metabolize vitamin D3 via this CYP11A1-mediated pathway that is modified by the activity of CYP27B1, with <em>20</em>(OH)D3 (the major metabolite), <em>20</em>,23(OH)2D3, 1,<em>20</em>(OH)2D3, 1,<em>20</em>,23(OH)3D3 and 17,<em>20</em>,23(OH)3D3 being detected, defining these secosteroids as endogenous regulators/natural products. This is supported by the detection of a mono-hydroxyvitamin D3 with the retention time of <em>20</em>(OH)D3 in human serum. In new work presented here we demonstrate that the CYP11A1-initiated pathways also occurs in Caco-2 colon cells. Our previous studies show that <em>20</em>(OH)D3 and <em>20</em>,23(OH)2D3 are non-calcemic at pharmacological doses, dependent in part on their lack of a C1α hydroxyl group. In epidermal keratinocytes, <em>20</em>(OH)D3, <em>20</em>(OH)D2 and <em>20</em>,23(OH)2D3 inhibited cell proliferation, stimulated differentiation and inhibited NF-κB activity with potencies comparable to 1,25(OH)2D3, acting as partial agonists on the VDR. 22(OH)D3 and <em>20</em>,22(OH)2D3, as well as secosteroids with a short or no side chain, showed antiproliferative and prodifferentiation effects, however, with lower potency than <em>20</em>(OH)D3 and <em>20</em>,23(OH)2D3. The CYP11A1-derived secosteroids also inhibited melanocyte proliferation while having no effect on melanogenesis, and showed anti-melanoma activities in terms of inhibiting proliferation and the ability to grow in soft agar. Furthermore, <em>20</em>(OH)D3 and <em>20</em>,23(OH)2D3 showed anti-fibrosing effects in vitro, and also in vivo for the former. New data presented here shows that <em>20</em>(OH)D3 inhibits LPS-induced production of TNFα in the J774 line, TNFα and <em>IL</em>-6 in peritoneal macrophages and suppresses the production of proinflammatory Th1/Th17-related cytokines, while promoting the production of the anti-inflammatory cytokine <em>IL</em>-10 in vivo. In summary, CYP11A1 initiates new pathways of vitamin D metabolism in a range of tissues and products could have important physiological roles at the local or systemic level. In the skin, CYP11A1-derived secosteroids could serve both as endogenous regulators of skin functions and as excellent candidates for treatment of hyperproliferative and inflammatory skin disorders, and skin cancer. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Due to their structural similarity, interleukin (<em>IL</em>)-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24 and <em>IL</em>-26 were combined with <em>IL</em>-10 in the so-called <em>IL</em>-10 family. To expand the knowledge on <em>IL</em>-19, <em>IL</em>-<em>20</em> and <em>IL</em>-24, we systematically and quantitatively analysed the expression of these mediators and their receptor chains in vitro and in vivo under various conditions and in comparison with other <em>IL</em>-10 family members. In vitro, <em>IL</em>-19, <em>IL</em>-<em>20</em> and <em>IL</em>-24 were produced not only by activated immune cells, particularly monocytes, but also to a similar extent by keratinocytes. <em>IL</em>-1beta increased the expression of these mediators 1000-fold (<em>IL</em>-19) and 10-fold (<em>IL</em>-<em>20</em> and <em>IL</em>-24) in keratinocytes. In vivo, these cytokines were expressed preferentially in inflamed tissues. The absence of either R1 chain for the two types of receptor complexes for these cytokines (<em>IL</em>-<em>20</em>R1/<em>IL</em>-<em>20</em>R2 and <em>IL</em>-22R1/<em>IL</em>-<em>20</em>R2) on immune cells implies that they cannot act on these cells. In fact, <em>IL</em>-19, <em>IL</em>-<em>20</em> and <em>IL</em>-24 did not induce activation of signal transducer and activator of transcription (STAT) molecules in immune cells. Instead, several tissues, particularly the skin, tissues from the reproductive and respiratory systems, and various glands appeared to be the main targets of these mediators. Keratinocytes expressed both receptor complexes; however, the expression of <em>IL</em>-22R1 was 10 times higher than that of <em>IL</em>-<em>20</em>R1. Interferon-gamma further increased the expression of <em>IL</em>-22R1 and decreased that of <em>IL</em>-<em>20</em>R1, suggesting that under T1 cytokine conditions these mediators primarily affect keratinocytes via the <em>IL</em>-22R1/<em>IL</em>-<em>20</em>R2 complex. In summary, these data support the notion that <em>IL</em>-19, <em>IL</em>-<em>20</em> and <em>IL</em>-24 are distinct from classical <em>ILs</em> and constitute a separate subfamily of mediators within the <em>IL</em>-10 family.

We investigated the effect of the angiotensin type 1 (AT-1) receptor antagonist, irbesartan, on matrix metalloproteinase (MMP) activity and cardiac cytokines in an animal model of diabetic cardiomyopathy. Diabetes was induced in <em>20</em> C57/bl6 mice by injection of streptozotocin (STZ). These animals were treated with irbesartan or placebo and were compared with nondiabetic controls. Left ventricular (LV) function was measured by pressure-volume loops with parameters for systolic function (end systolic elastance [Ees]) and diastolic function (cardiac stiffness) 8 weeks after STZ treatment. The cardiac protein content of interleukin (<em>IL</em>)1beta and transforming growth factor (TGF)beta1 were measured by enzyme-linked immunosorbent assay. The total cardiac collagen content and collagen type 1 and 3 were measured by histochemistry, and MMP-2 activity was measured by gelatin zymography. LV dysfunction was documented by impaired Ees and diastolic stiffness in STZ mice compared with controls. This was accompanied by increased TGFbeta, <em>IL</em>1beta, and fibrosis and decreased MMP-2 activity. Treatment with irbesartan attenuated LV dysfunction, <em>IL</em>1beta, TGFbeta, and cardiac fibrosis compared with untreated diabetic animals and normalized MMP activity. These findings present evidence that AT-1 receptor antagonists attenuate cardiac failure by decreasing cardiac inflammation and normalizing MMP activity, leading to normalized cardiac fibrosis in STZ-induced diabetic cardiomyopathy.

OBJECTIVE

This study examined the effects of darapladib, a selective lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) inhibitor, on biomarkers of cardiovascular (CV) risk.

BACKGROUND

Elevated Lp-PLA(2) levels are associated with an increased risk of CV events.

METHODS

Coronary heart disease (CHD) and CHD-risk equivalent patients (n = 959) receiving atorvastatin (<em>20</em> or 80 mg) were randomized to oral darapladib 40 mg, 80 mg, 160 mg, or placebo once daily for 12 weeks. Blood samples were analyzed for Lp-PLA(2) activity and other biomarkers.

RESULTS

Baseline low-density lipoprotein cholesterol (LDL-C) was 67 +/- 22 mg/dl. Plasma Lp-PLA(2) was higher in older patients >>or=75 years), in men, in those taking atorvastatin <em>20</em> mg, at LDL-C>>or=70 mg/dl or high-density lipoprotein cholesterol (HDL-C) <40 mg/dl, or in those with documented vascular disease (multivariate regression; p < 0.01). Darapladib 40, 80, and 160 mg inhibited Lp-PLA(2) activity by approximately 43%, 55%, and 66% compared with placebo (p < 0.001 weeks 4 and 12). Sustained dose-dependent inhibition was noted overall in both atorvastatin groups and at different baseline LDL-C >>or=70 vs. <70 mg/dl) and HDL-C (<40 vs.>>or=40 mg/dl). At 12 weeks, darapladib 160 mg decreased interleukin (IL)-6 by 12.3% (95% confidence interval [CI] -22% to -1%; p = 0.028) and high-sensitivity C-reactive protein (hs-CRP) by 13.0% (95% CI -28% to +5%; p = 0.15) compared with placebo. The Lp-PLA(2) inhibition produced no detrimental effects on platelet biomarkers (P-selectin, CD40 ligand, urinary 11-dehydrothromboxane B(2)). No major safety concerns were noted.

CONCLUSIONS

Darapladib produced sustained inhibition of plasma Lp-PLA(2) activity in patients receiving intensive atorvastatin therapy. Changes in IL-6 and hs-CRP after 12 weeks of darapladib 160 mg suggest a possible reduction in inflammatory burden. Further studies will determine whether Lp-PLA(2) inhibition is associated with favorable effects on CV events.

BACKGROUND

IL-17 and Foxp3 double-expressing (DE) CD4(+) T lymphocytes are novel crossover immune cell population, but the presence and role of these cells in human intestinal inflammation is unclear. The aim of this study was to investigate the circulating IL-17 and Foxp3 DE CD4(+) T lymphocytes in patients with inflammatory bowel disease (IBD).

METHODS

The entire cohort consisted of 79 subjects: 31 patients with Crohn's disease, 28 patients with ulcerative colitis, and 20 healthy control subjects (HC). IBD patients with evidence of active disease at endoscopy were entered into the study. Peripheral blood mononuclear cells were used for ex vivo and in vitro studies to assess the characteristics and generation of these novel cells and the function of circulating Foxp3 CD4(+) regulatory T lymphocytes (Treg) in patients with IBD compared with HC.

RESULTS

Patients with IBD had significantly higher prevalence of IL-17 and Foxp3 DE CD4(+) T lymphocytes compared with age- and gender-matched HC. These cells expressed RORγt. The ability of Treg cells to suppress autologous T-cell proliferation was reduced by approximately 60% in patients with IBD compared with HC. Increased generation of these DE cells was demonstrated by the modulation of cytokine environment of CD4(+) lymphocytes in vitro in patients with Crohn's disease.

CONCLUSIONS

Prevalence of circulating IL-17 and Foxp3 DE CD4(+) T cells is increased in patients with IBD. Coexpression of RORγt and Foxp3 in these cells implies conversion from Treg cells to Th17 cells. This is associated with a decreased suppressive function of Foxp3 CD4(+) T lymphocytes in patients with IBD.

Focal cortical dysplasia (FCD) and glioneuronal tumors (GNT) are recognized causes of chronic intractable epilepsy. The cellular mechanism(s) underlying their epileptogenicity remain largely unknown. Compelling evidence in experimental models of seizures indicates an important role of interleukin (<em>IL</em>)-1beta in the mechanisms of hyperexcitability leading to the occurrence of seizures. We immunocytochemically investigated the brain expression and cellular distribution pattern of <em>IL</em>-1beta, <em>IL</em>-1 receptor (<em>IL</em>-1R) types I and II and <em>IL</em>-1R antagonist (<em>IL</em>-1Ra) in FCD and GNT specimens, and we correlate these parameters with the clinical history of epilepsy in patients with medically intractable seizures. In normal control cortex, and in perilesional regions with histologically normal cortex, <em>IL</em>-1beta, <em>IL</em>-1Rs and <em>IL</em>-1Ra expression was undetectable. In all FCD and GNT specimens, <em>IL</em>-1beta and its signalling receptor <em>IL</em>-1RI were highly expressed by more than 30% of neurons and glia whereas the decoy receptor <em>IL</em>-RII and <em>IL</em>-Ra were expressed to a lesser extent by approximately 10% and <em>20</em>% of cells, respectively. These findings show a high expression of <em>IL</em>-1beta and its functional receptor (<em>IL</em>-1RI) in FCD and GNT specimens together with a relative paucity of mechanisms (<em>IL</em>-1RII and <em>IL</em>-1Ra) apt to inactivate <em>IL</em>-1beta actions. Moreover, the number of <em>IL</em>-1beta- and <em>IL</em>-1RI-positive neurons was positively correlated with the frequency of seizures, whereas the number of <em>IL</em>-1Ra-positive neurons and astroglial cells was negatively correlated with the duration of epilepsy prior to surgery. The expression of <em>IL</em>-1beta family members in these developmental lesions may contribute to their intrinsic and high epileptogenicity, thus possibly representing a novel target for antiepileptic strategies.

OBJECTIVE

This study examined the effect of statin therapy on vascular markers of inflammation and echocardiographic findings in patients with nonischemic forms of cardiomyopathy.

BACKGROUND

Despite advances in therapy, morbidity and mortality from heart failure (HF) remain high. We wished to determine whether treatment with atorvastatin affects left ventricular (LV) systolic function and markers of inflammation in patients with nonischemic HF.

METHODS

A total of 108 patients with nonischemic HF and a left ventricular ejection fraction (LVEF) < or =35% were randomized to either atorvastatin <em>20</em> mg/day or placebo in a double-blinded fashion for a 12-month period. The LVEF and LV end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were determined by echocardiography. Serum markers of inflammation and oxidation were also measured.

RESULTS

The LVEF increased from 0.33 +/- 0.05 to 0.37 +/- 0.04 (p = 0.01) in the atorvastatin group over the 12-month follow-up period, whereas those patients in the placebo group experienced a decline in ejection fraction during the same time period. In addition, LVEDD was reduced from 57.1 +/- 5.9 mm to 53.4 +/- 5.1 mm (p = 0.007) and LVESD was reduced from 42.4 +/- 3.8 mm to 39.1 +/- 3.8 mm (p = 0.02) in the cohort of patients treated with atorvastatin; these dimensions increased in the placebo group. There was an increase in erythrocyte superoxide dismutase (E-SOD) activity, and there were significant reductions in serum levels of high sensitivity C-reactive protein, interleukin-6 (IL-6), and tumor necrosis factor-alpha receptor II (TNF-alpha RII) in the atorvastatin group.

CONCLUSIONS

The use of atorvastatin in patients with nonischemic HF improves LVEF and attenuates adverse LV remodeling. The effects on soluble levels of several inflammatory markers with atorvastatin suggest, in part, mechanisms by which statins might exert their beneficial effects in nonischemic HF.

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The Apc(Min/+) mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating <em>IL</em>-6. However, the <em>IL</em>-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the Apc(Min/+) mouse is not known. Cachexia progression was studied in Apc(Min/+) mice that were either weight stable (WS) or had initial (≤5%), intermediate (6-19%), or extreme (≥<em>20</em>%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. <em>IL</em>-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. <em>IL</em>-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of <em>IL</em>-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.

The incidence and mortality of sepsis increase with age, consequently, 80% of the clinical mortality from sepsis occurs in patients over age 65. Despite this aged clinical population, most research models of sepsis use 6- to 16-week-old mice as patient surrogates. This age range of mice corresponds to human ages 10 to 17 years. To assess the influence of age on rodent CLP and on antibiotic therapy, we studied young (4 month), mature (12 month), and aged (24 month) mice. Male C57BL/6 mice (n = 27-30 in each age group) were subjected to cecal ligation and puncture (CLP), two punctures with a 25-gauge needle. Mice were observed untreated for 10 days. Young mice had <em>20</em>% mortality, mature mice had 70% mortality (P = 0.0013 vs. young), and aged mice had 75% mortality (P = 0.0001 vs. young). To assess the effects of age on antibiotic therapy, mice were subjected to CLP as above (n = 38-40 in each age group). Mice were then randomized to treatment with intraperitoneal injections of ceftriaxone and metronidazole or normal saline. Therapy was initiated 12 h after CLP, and injections were repeated every 12 h for 7 days. Young mice saw a 56% decrease in mortality from CLP with antibiotic therapy (P = 0.001), and mature mice had a 30% decrease in mortality (P = 0.06). Aged mice saw no benefit from antibiotic therapy. We also compared plasma cytokine levels between young and aged mice after CLP. When compared with young mice, aged mice had higher levels of <em>IL</em>-6 and TNF-alpha 24 h after CLP. However, high <em>IL</em>-6 was predictive of mortality at any age. Mice appear to have age-dependent responses to intra-abdominal sepsis and to appropriate therapy.

BACKGROUND

Regulatory T (Treg) cells play an important role in controlling allergic inflammation. The transcription factor Foxp3 regulates the development and function of natural and adaptive CD4(+)CD25(+) Treg cells.

OBJECTIVE

We sought to examine the effect of grass pollen injection immunotherapy on the numbers of Foxp3(+)CD4(+) and Foxp3(+)CD25(+) T cells in and out of season and their expression of IL-10 in the nasal mucosa of patients with hay fever.

METHODS

Nasal biopsy specimens were obtained from untreated patients with hay fever, participants with grass pollen allergy who had received 2 years of immunotherapy, and healthy control subjects. Dual-immunofluorescence microscopy was used to enumerate and colocalize Foxp3 expression to CD4(+) and CD25(+) T cells in the nasal mucosa. Triple staining was performed to colocalize Foxp3(+) cells to CD3(+)CD25(+) and CD3(+) IL-10-expressing cells.

RESULTS

At peak season, numbers of Foxp3(+)CD25(+) (P = .02) and Foxp3(+)CD4(+) (P = .03) cells were significantly increased in the nasal mucosa of immunotherapy-treated patients compared with numbers before treatment. Foxp3(+)CD25(+) (P = .03) and Foxp3(+)CD4(+) (P = .04) cells were also greater in immunotherapy-treated patients out of season compared with those in untreated patients with hay fever. Within the immunotherapy-treated group, 20% of CD3(+)CD25(+) cells expressed Foxp3, and 18% of Foxp3(+)CD3(+) cells were IL-10 positive.

CONCLUSIONS

The presence of local Foxp3(+)CD25(+)CD3(+) cells in the nasal mucosa, their increased numbers after immunotherapy, and their association with clinical efficacy and suppression of seasonal allergic inflammation support a putative role for Treg cells in the induction of allergen-specific tolerance in human subjects.

Psychological stress can lead to asthma exacerbations in some patients. It is our hypothesis that the stress effect can occur through an enhancement of allergic inflammatory response. To investigate this possibility, airway antigen challenge was evaluated in <em>20</em> college students with mild asthma during both a low-stress phase (midsemester or two weeks postfinal examination) and a stress phase (final examination week). Subjects completed questionnaires to assess psychological state and underwent inhaled antigen challenge. Sputum samples were collected before challenge, and six and 24 hours and seven days postchallenge. Leukocytes were counted and eosinophil-derived neurotoxin (EDN) was measured in sputum supernates. Sputum cells were cultured and stimulated ex vivo with phytohemagglutinin (10 microg/ml), and culture supernates were assayed for interleukin-5 (<em>IL</em>-5) and interferon-gamma by enzyme-linked immunosorbent assay. Sputum eosinophils and EDN levels significantly increased at six and 24 hours postchallenge and were enhanced during the stress phase (p < 0.01). <em>IL</em>-5 generation by sputum cells was also increased at 24 hours during stress and correlated with airway eosinophils (r(s) = 0.65, p < 0.05). Students' anxiety and depression scores were significantly higher during the examination period. Our findings suggest that stress associated with final examinations can act as a cofactor to increase eosinophilic airway inflammation to antigen challenge and thus may enhance asthma severity.

In this report a method for the affinity purification and radiolabeling of recombinant mouse interleukin (<em>IL</em>)-4 is described. It is shown on the basis of several criteria that <em>IL</em>-4 retains full biologic activity after radioiodination and can therefore be used as a valid model for measuring the binding characteristics of native <em>IL</em>-4. By using Scatchard plot analysis of equilibrium binding data, it is demonstrated that 125I-<em>IL</em>-4 binds to a high affinity cell surface receptor which is expressed by both hemopoietic and nonhemopoietic cells. The dissociation constant for 125I-<em>IL</em>-4 (Kd = <em>20</em> to 60 pM) corresponds to the concentration of <em>IL</em>-4 which gives 50% biologic activity (i.e., 10 to 30 pM). Binding of 125I-<em>IL</em>-4 is rapid (t1/2 of 2 min), whereas dissociation occurs at a slow rate (t1/2 approximately 4 hr). The <em>IL</em>-4 receptor shows a high degree of specificity. Whereas unlabeled mouse <em>IL</em>-4 competed with mouse 125I-<em>IL</em>-4 in an equimolar fashion for binding to <em>IL</em>-4 receptors, several other lymphokines, including mouse <em>IL</em>-2, <em>IL</em>-3, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and human <em>IL</em>-1, <em>IL</em>-2, and <em>IL</em>-4 were unable to inhibit, even at molar excesses of 400 to 800-fold. At 37 degrees C, 125I-<em>IL</em>-4 is rapidly internalized (approximately <em>20</em>0 molecules/cell/min) by HT-2 cells, with at least 85% of cell surface receptors being functional in this respect. Receptors for <em>IL</em>-4 were found to be expressed by subclasses of T and B cells, mast cells, macrophages, and by cells of the myeloid and erythroid lineages. This wide distribution of receptor expression closely matches the known spectrum of biologic activities of <em>IL</em>-4, including proliferation and/or differentiation of T and B cells, mast cells and granulocytes, and induction of macrophage antigen-presenting capacity. <em>IL</em>-4 receptors were also found on a variety of nonhemopoietic cells such as cloned stromal cell lines from the bone marrow, spleen, thymus, and brain, and on muscle, brain, melanoma, fibroblast, and liver cells. Indeed, only 5 of more than 90 cell types tested have undetectable numbers of <em>IL</em>-4 receptors. The biologic effects of <em>IL</em>-4 on nonhemopoietic cells have not yet been reported and await elucidation.

Adherent human mesangial cells (HMC) were unable to phagocytose serum-treated zymosan (STZ), nevertheless this stimulus (1 mg/ml) induced a marked immediate increase of H2O2 and O2- release at a rate of 3.15 +/- 0.35 and 3.40 +/- 0.12 nmol/10(6) HMC/hr, respectively. Zymosan alone resulted in no release of either H2O2 or O2-. Phorbol myristate acetate (PMA, 2 X 10(-6) M) had only marginal effects on HMC leading to the generation of 0.273 +/- 0.014 nmol O2-/10(6) HMC/hr. After a lag period, human recombinant tumor necrosis factor-alpha (TNF-alpha) and human recombinant interleukin 1-alpha <em>IL</em>-1 alpha) both induced significant O2- production measured as SOD inhibitable reduction of cytochrome c, 5 X 10(-5) M, by adherent HMC for up to five hours, the maximum rates being 3.04 +/- 0.08 and 3.2 +/- 0.08 nmol/10(6) HMC/hr for <em>IL</em>-1 alpha and TNF-alpha, respectively. Significant O2- release was detectable at 0.625 ng/ml (37 pM) <em>IL</em>-1 alpha or 1 ng/ml (59 pM) TNF-alpha (P less than 0.05). Catalase inhibitable H2O2 production was also induced by <em>IL</em>-1 alpha and TNF-alpha in a dose dependent manner. Using scopoletin (40 nM) and 1 microM peroxidase we fluorimetrically measured 1.73 +/- 0.14 and 1.49 +/- 0.19 nmol H2O2/10(6) HMC/hr induced by <em>IL</em>-1 alpha (25 ng/ml) and TNF-alpha (<em>20</em> ng/ml). Finally, we ascertained the type of radical species produced by HMC stimulated by cytokines employing ESR-spin-trapping with DMPO.2+ These results demonstrated that O2- was the primary radical species formed.(ABSTRACT TRUNCATED AT 250 WORDS)

CD11/CD18 and VLA-4 integrins mediate interactions of monocytes with HUVEC cultured on human amniotic tissue. In the present study, the roles of individual CD11/CD18 integrins and endothelial adhesion molecules were examined using blocking mAbs and peptides. After <em>20</em> min of incubation, monocyte adhesion to and migration across unstimulated endothelium was dependent primarily on CD11a/CD18. When incubation was extended to 2 h to allow for completion of migration, either CD11a/CD18 or CD11b/CD18 could be used. Similarly, either CD11a/CD18 or CD11b/CD18 could be used by monocytes to bind to and traverse <em>IL</em>-1 beta-stimulated endothelium. Although both CD11a/CD18 and CD11b/CD18 are known to bind to ICAM-1, results of Ab-mixing experiments suggest that alternative ligands on HUVEC for CD11/CD18 integrins also may be used during transendothelial migration of monocytes. Our previous studies indicate that VLA-4 on monocytes interacts primarily with VCAM-1 on unstimulated endothelium. In contrast, migration of monocytes across <em>IL</em>-1 beta-stimulated endothelium was less dependent on VCAM-1. mAbs directed against binding sites for VLA-4 in domain 1 and domain 4 of VCAM-1 did not, by themselves, inhibit interactions of monocytes with stimulated HUVEC. VLA-4-dependent migration across <em>IL</em>-1 beta-stimulated endothelium was markedly inhibited only when mAbs to VCAM-1 were added in combination with peptides of fibronectin. Therefore, VLA-4 can interact with either VCAM-1 or alternative ligands on <em>IL</em>-1 beta-stimulated HUVEC-amnion cultures to mediate transendothelial migration of monocytes.

We previously showed that endothelial cells (EC) from the vasculature of human solid tumors have a decreased expression of intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 as compared with normal tissue EC. This effect is explained by EC exposure to angiogenic factors. It is known that upregulation of endothelial adhesion molecules (EAM) is a sign of EC activation in inflammatory responses. We therefore tested the effect of angiogenic factors on upregulation of EAM on tumor EC and human umbilical vein EC (HUVEC) by proinflammatory cytokines. Incubation of tumor-derived EC in tumor necrosis factor alpha (TNF alpha) did result in expression levels of only <em>20</em>% of the level of similarly treated normal tissue-derived EC. Pretreatment of HUVEC with 10 ng/ml basic fibroblast growth factor (bFGF) for 3 days, before TNF alpha- or interleukin-1 alpha (<em>IL</em>-1 alpha) stimulation, resulted in ICAM-1 levels of only 30% to 60% of cells without pretreatment. Also, the induction of vascular EC adhesion molecule-1 (VCAM-1) and E-selectin by TNF alpha was significantly inhibited by prior exposure to bFGF. Vascular endothelial growth factor had similar but less prominent effects. The effect of transforming growth factor-beta and <em>IL</em>-8 was studied as well. The functional relevance of the finding of a decreased EC inflammatory response was confirmed by adhesion assays. Our results show that tumor angiogenesis induces EC anergy. This may serve as a tumor-protecting mechanism by impairing the development of an efficient leukocyte infiltrate in tumors.

The contribution of exogenous and endogenous TGF-beta 1 to human peripheral blood NK cell proliferation and activity in vitro was investigated. Exogenous bioactive TGF-beta 1 inhibited NK cell DNA synthesis and production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) by NK cultures consisting of>> 98% CD56+ cells. The cytotoxic activity of NK cells was also weakly inhibited by exogenous TGF-beta 1. All TGF-beta 1-induced inhibitory effects occurred in the absence and presence of the NK cell-activating cytokines IFN-alpha, <em>IL</em>-2, and <em>IL</em>-12. Unstimulated NK cell cultures expressed steady state TGF-beta 1 mRNA detected by Northern blot analysis and produced TGF-beta protein (1.6 ng/ml), as determined by ELISA. When NK cell proliferation was induced by <em>IL</em>-2, <em>IL</em>-12, IFN-alpha, or a combination of <em>IL</em>-2 and <em>IL</em>-12, expression of TGF-beta 1 mRNA and protein was moderately and consistently reduced by approximately <em>20</em>%, as compared with unstimulated control cultures. Unstimulated and rapidly proliferating NK cell cultures secreted primarily latent TGF-beta into their culture medium, as determined by the Mv1Lu bioassay. These results indicate that, in vitro, endogenous NK cell-derived TGF-beta 1 has no negative autocrine effect upon activation of NK cells by various cytokines.

T(h)17 cells represent a new pro-inflammatory T(h) cell lineage distinct from T(h)1 and T(h)2 cells. T(h)17 cells have been shown to be involved in extracellular bacterial infection but their role in intracellular infection remains unclear. We found antigen-specific <em>IL</em>-17A production during a systemic infection of mice with the facultative intracellular bacterium Salmonella enterica serovar Enteritidis (S. Enteritidis) and examined the function and cellular source of <em>IL</em>-17A during the adaptive immune response to S. Enteritidis. Infected <em>IL</em>-17A-/- mice survived completely after inoculation with the highest infection dose found to be sub-lethal for wild-type (WT) C57BL/6 mice. However, at <em>20</em> and 80 days post-infection (d.p.i.), we repeatedly found mildly elevated bacterial burden in spleen and liver of <em>IL</em>-17A-/- mice as compared with WT mice. Overall, <em>IL</em>-17A-/- mice showed reduced clearance of S. Enteritidis. S. Enteritidis-specific <em>IL</em>-17A production was induced in splenocytes and lymph node cells of infected WT mice at both time points, <em>20</em> and 80 d.p.i. Classical CD4+ T(h)17 cells developed upon infection with Salmonella. CD4- gammadelta TCR+ and CD4- gammadelta TCR- cells were found to be additional <em>IL</em>-17A-producing cell populations. In infected <em>IL</em>-17A-/- mice, a normal T(h)1 cytokine profile was observed consistent with the overall subtle phenotype. Nevertheless, in the absence of <em>IL</em>-17A, recruitment of neutrophils and delayed-type hypersensitivity (DTH) reactivity was significantly compromised. Our data indicate that <em>IL</em>-17A responses are induced by Salmonella and mildly contribute to protective immunity during S. Enteritidis infection. Thus, <em>IL</em>-17A complements the <em>IL</em>-12/IFN-gamma axis which is essential for protective immunity against salmonellosis in mice and men.