Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1-10 micrometer polymers of N-acetyl-<cmd SC>d<cmd /SC> -glucosamine) through mannose receptors and produce <em>IL</em>-12, <em>IL</em>-18, and TNF-alpha. These cytokines lead to the production of IFN-gamma by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 x 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of <em>IL</em>-4 (0.6 ng/ml), <em>IL</em>-5 (<em>20</em> U/ml), and <em>IL</em>-10 (3.2 ng/ml), but not IFN-gamma. Ragweed/chitin stimulation resulted in significant decreases of <em>IL</em>-4, <em>IL</em>-5, and <em>IL</em>-10 levels and the production of IFN-gamma (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-gamma production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.

Tuberculosis has emerged as an epidemic fueled by the large number of individuals infected with the human immunodeficiency virus, especially those who are injecting drug users. We found a striking increase from 4- to <em>20</em>8-fold in p24 levels in bronchoalveolar lavage fluid from involved sites of Mycobacterium tuberculosis infection vs uninvolved sites in three HIV+ patients. We used an in vitro cell culture model to determine if tuberculosis could activate replication of HIV-1. Mononuclear phagocyte cell lines U937 and THP-1 infected with HIV-1JR-CSF, in vitro and stimulated with live M. tuberculosis H37Ra, had a threefold increase in p24 in culture supernatants. Using the HIV-1 long terminal repeat with a chloramphenicol acetyltransferase (CAT) reporter construct, live M. tuberculosis increased transcription <em>20</em>-fold in THP-1 cells, and cell wall components stimulated CAT expression to a lesser extent. The nuclear factor-kappa B enhancer element was responsible for the majority of the increased CAT activity although two upstream nuclear factor-<em>IL</em>6 sites may also contribute to enhanced transcription. Antibodies to TNF-alpha and <em>IL</em>-1 inhibited the increase in CAT activity of the HIV-1 long terminal repeat by M. tuberculosis from 21-fold to 8-fold. Stimulation of HIV-1 replication by M. tuberculosis may exacerbate dysfunction of the host immune response in dually infected individuals.

The present study was undertaken to test the hypothesis that tumor necrosis factor (TNF) and/or interleukin-1 (<em>IL</em>-1) activity mediates lipopolysaccharide (LPS)-induced bone resorption in vivo. To test this hypothesis, Escherichia coli LPS or Porphyromonas gingivalis LPS was injected into the subcutaneous tissues overlying mouse calvariae. Histological sections, prepared from the center of the lesion, were stained for tartrate-resistant acid phosphatase, and histomorphometric analysis was performed to quantify the osteoclast number and the area of bone resorption. In time course experiments using normal mice, a peak of bone resorption occurred 5 days after endotoxin stimulation. In dose-response experiments, <em>IL</em>-1 receptor type 1 deletion (<em>IL</em>-1R(-/-)), TNF double-receptor p55/p75 deletion (TNF p55(-/-)/p75(-/-)), combined TNF p55 and <em>IL</em>-1 receptor type 1 deletion (TNF p55(-/-)/<em>IL</em>-1R(-/-)), and <em>IL</em>-1beta-converting enzyme-deficient (ICE(-/-)) mice and the respective wild-type mice were injected with 500, 100, or <em>20</em> micrograms of P. gingivalis LPS and sacrificed 5 days after LPS injection. At the highest dose (500 micrograms), significant decreases in osteoclast number occurred in mutant mice compared to wild-type mice: (i) a 64% reduction for the TNF p55(-/-)/<em>IL</em>-1R(-/-) mice, (ii) a 57% reduction for the <em>IL</em>-1R(-/-) mice, (iii) a 41% reduction for the TNF p55(-/-)/p75(-/-) mice, and (iv) a 38% reduction for the ICE(-/-) mice. At the two lower doses, bone resorption was apparent but no significant differences between mutant and wild-type animals were observed. The present data indicate that at higher doses, LPS-induced bone resorption is substantially mediated by <em>IL</em>-1 and TNF receptor signaling. Furthermore, <em>IL</em>-1 receptor signaling appears to be slightly more important than TNF receptor signaling. At lower LPS doses, other pathways leading to osteoclast activity that are independent of TNF and <em>IL</em>-1 are involved.

BACKGROUND

Previous studies suggested miR-146a and miR-155 play important roles in innate and adaptive immune responses. We studied intra-renal and urinary levels of miR-146a and miR-155 in patients with immunoglobulin A nephropathy (IgAN).

METHODS

Intra-renal and urinary levels of miR-146a and miR-155 are quantified in 43 patients with IgAN; the result was compared to <em>20</em> nephrectomy specimens and urine sediment of 13 healthy volunteers.

RESULTS

The levels of intra-renal and urinary levels of miR-146a and miR-155 of IgAN are significantly higher than controls. Estimated glomerular filtration rate inversely correlates with intra-renal level of miR-146a and miR-155; proteinuria positively correlates with intra-renal level of miR-146a and miR-155, as well as urinary level of miR-146a and miR-155. Intra-renal level of miR-155 significantly correlates with tubulointerstitial scarring. Urinary level of miR-146a inversely correlates with urinary expression of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α and positively correlates with urinary expression of regulated upon activation, normal T-cell expressed, and secreted (RANTES). Urinary level of miR-155 inversely correlates with urinary expression of IL-1β and TNF-α and positively correlates with urinary expression of forkhead box P3 (FOXP3) and RANTES.

CONCLUSIONS

We conclude that intra-renal and urinary levels of miR-146a and miR-155 were significantly elevated in IgAN, and the degree of upregulation correlates with clinical and histological severity of the disease. Our results suggested miR-146a and miR-155 might play an important role in the pathophysiology of IgAN.

OBJECTIVE

Leptin, an adipocyte-secreted hormone, has emerged as a potential candidate for the link between obesity and the proinflammatory state. Specifically, leptin modulates T-helper (Th) cells toward a Th1 phenotype, with the secretion of proinflammatory cytokines. The aim of this study was to evaluate the Th1/Th2 balance in obese children and its relation with hormonal and metabolic features.

METHODS

In 50 obese children and <em>20</em> control children, we measured the CD4-positive Th cells that secrete interferon (IFN)-gamma or interleukin (<em>IL</em>)-2 (taken as an index of Th1 cells), and <em>IL</em>-4 (taken as an index of Th2 cells) as well as serum glucose, insulin, insulin resistance (IR) index (as homeostasis model assessment model (HOMA)), lipid profile, aminotransferases, leptin and ghrelin. Obese children also underwent dual energy X-ray absorptiometry scan measurements, and liver ultrasound scanning.

RESULTS

Geometric mean percentages of IL-2- and IL-4-CD4 secreting cells in obese children were not significantly different from those found in control children. However, the geometric mean percentage of CD4-positive T cells secreting IFN-gamma was significantly higher in the obese than in the control (P < 0.0001, t-test) group. Within the entire group of study children, the percentage of IFN-gamma-positive cells was positively associated with leptin (P = 0.002), insulin (P < 0.00 005), and HOMA-IR values (P < 0.00 005). However, when these associations were restricted to the group of obese subjects, insulin and HOMA-IR values, but not leptin, retained statistical significance. Yet, in the obese group, the percentage of IFN-gamma-positive cells was associated with nonalcoholic steatohepatitis (NASH) (P = 0.001), but not with body mass index-standard deviation score and total body fat mass.

CONCLUSIONS

In obese children, a shift to Th1-cytokine profile dominated by the production of IFN-gamma is related to insulin resistance as well as to NASH independently of anthropometric features and other metabolic characteristics. The prevalent Th1 pattern of secreted cytokines may be regarded as a mechanism contributing to inflammation in obesity.

<em>IL</em>-22 is an <em>IL</em>-10 family cytokine that initiates innate immune responses against bacterial pathogens and contributes to immune disease. <em>IL</em>-22 biological activity is initiated by binding to a cell-surface complex composed of <em>IL</em>-22R1 and <em>IL</em>-10R2 receptor chains and further regulated by interactions with a soluble binding protein, <em>IL</em>-22BP, which shares sequence similarity with an extracellular region of <em>IL</em>-22R1 (s<em>IL</em>-22R1). <em>IL</em>-22R1 also pairs with the <em>IL</em>-<em>20</em>R2 chain to induce <em>IL</em>-<em>20</em> and <em>IL</em>-24 signaling. To define the molecular basis of these diverse interactions, we have determined the structure of the <em>IL</em>-22/s<em>IL</em>-22R1 complex. The structure, combined with homology modeling and surface plasmon resonance studies, defines the molecular basis for the distinct affinities and specificities of <em>IL</em>-22 and <em>IL</em>-10 receptor chains that regulate cellular targeting and signal transduction to elicit effective immune responses.

BACKGROUND

Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective.

METHODS

Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = <em>20</em>) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, <em>IL</em>-6 and C-reactive protein (CRP).

RESULTS

Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient.

CONCLUSIONS

Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.

OBJECTIVE

Proinflammatory cytokines in degenerative diseases can lead to the loss of normal physiology and the destruction of surrounding tissues. In the present study, the physiological responses of human fetal retinal pigment epithelia (hfRPE) were examined in vitro after polarized activation of proinflammatory cytokine receptors.

METHODS

Primary cultures of hfRPE were stimulated with an inflammatory cytokine mixture (ICM): interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. Western blot analysis and immunofluorescence were used to determine the expression/localization of the cytokine receptors on hfRPE. Polarized secretion of cytokines was measured. A capacitance probe technique was used to measure transepithelial fluid flow (J(V)) and resistance (R(T)).

RESULTS

IL-1R1 was mainly localized to the apical membrane and TNFR1 to the basal membrane, whereas IFN-gammaR1 was detected on both membranes. Activation by apical ICM induced a significant secretion of angiogenic and angiostatic chemokines, mainly across the hfRPE apical membrane. Addition of the ICM to the basal but not the apical bath significantly increased net fluid absorption (J(V)) across the hfRPE within 20 minutes. Similar increases in J(V) were produced by a 24-hour exposure to ICM, which significantly decreased total R(T).

CONCLUSIONS

Chemokine gradients across the RPE can be altered (1) through an ICM-induced change in polarized chemokine secretion and (2) through an increase in ICM-induced net fluid absorption. In vivo, both of these factors could contribute to the development of chemokine gradients that help mediate the progression of inflammation/angiogenesis at the retina/RPE/choroid complex.

Psoriasis is a common chronic skin disease. Recent studies demonstrated that <em>IL</em>-<em>20</em> and <em>IL</em>-22, cytokines produced by keratinocytes and T cells, respectively, both inhibit keratinocyte terminal differentiation and induce psoriasis-like epidermis alterations. Here, we investigated the relationship between these mediators. Although <em>IL</em>-<em>20</em> was not able to regulate <em>IL</em>-22 production, <em>IL</em>-22 induced <em>IL</em>-<em>20</em> mRNA and protein in human keratinocytes. However, <em>IL</em>-22 had only a minimal effect, if any, on <em>IL</em>-19 and <em>IL</em>-26. Cutaneous <em>IL</em>-<em>20</em> was also elevated in mice following <em>IL</em>-22 application. Accordingly, some of <em>IL</em>-22's effects on differentiation-regulating genes were partially mediated by an endogenous, secreted protein and attenuated by anti-<em>IL</em>-<em>20</em> Ab. Like <em>IL</em>-22, <em>IL</em>-17A and TNF-alpha induced <em>IL</em>-<em>20</em> in keratinocytes, whereas IFN-gamma and <em>IL</em>-<em>20</em> itself did not. Furthermore, <em>IL</em>-17A and TNF-alpha individually strengthened the <em>IL</em>-22-induced <em>IL</em>-<em>20</em> production. In lesional skin of psoriasis patients, highly elevated <em>IL</em>-<em>20</em> levels strongly correlated with <em>IL</em>-22, and to a lesser extent, with <em>IL</em>-17A and TNF-alpha. As previously shown for <em>IL</em>-22, <em>IL</em>-<em>20</em> blood levels correlated with the disease severity, although with a lower significance. This study demonstrates that a T-cell mediator induces a tissue cell mediator with similar effects to its own and therefore suggests the existence of a novel type of pathogenetic cascade.

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast's gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of <em>IL</em>-1beta and a twofold greater <em>IL</em>-1beta-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive <em>IL</em>-1beta. Most notably, the <em>20</em>-fold induction of <em>IL</em>-6 mRNA and protein was completely blocked by <em>IL</em>-1 receptor antagonist. The combined addition of <em>IL</em>-1beta and <em>IL</em>-6 was mitogenic, and the proliferative response to conditioned medium from <em>IL</em>-1beta-exposed cells was blocked by antagonistically acting Abs to <em>IL</em>-6 or to gp130. These novel findings indicate that soluble <em>IL</em>-1beta bioactivity and autocrine <em>IL</em>-1beta-dependent <em>IL</em>-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.

<em>IL</em>-12, like <em>IL</em>-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither <em>IL</em>-12 nor <em>IL</em>-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that <em>IL</em>-12, like <em>IL</em>-18, was unable to act directly on osteoclastic precursors. <em>IL</em>-12, like <em>IL</em>-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of <em>IL</em>-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted <em>IL</em>-12 or <em>IL</em>-18 to be inhibitory. Additionally, <em>IL</em>-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, <em>IL</em>-18 and <em>IL</em>-12 synergistically inhibited osteoclast formation at concentrations <em>20</em>- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to <em>IL</em>-4, <em>IL</em>-10, or <em>IL</em>-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to <em>IL</em>-12 and <em>IL</em>-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.

OBJECTIVE

Our objective was to determine whether exenatide exerts an antiinflammatory effect.

METHODS

Twenty-four patients were prospectively randomized to be injected sc with either exenatide 10 μg twice daily [n = 12; mean age = 56 ± 3 yr; mean body mass index = 39.8 ± 2 kg/m(2); mean glycosylated hemoglobin (HbA1c) = 8.6 ± 0.4%] or placebo twice daily (n = 12; mean age = 54 ± 4 yr; mean body mass index = 39.1 ± 1.6 kg/m(2); mean HbA1c = 8.5 ± 0.3%) for 12 wk. Fasting blood samples were obtained at 0, 3, 6, and 12 wk. Blood samples were also collected for up to 6 h after a single dose of exenatide (5 μg) or placebo.

RESULTS

Fasting blood glucose fell from 139 ± 17 to 110 ± 9 mg/dl, HbA1c from 8.6 ± 0.4 to 7.4 ± 0.5% (P < 0.05), and free fatty acids by 21 ± 5% from baseline (P < 0.05) with exenatide. There was no weight loss. There was a significant reduction in reactive oxygen species generation and nuclear factor-κB binding by 22 ± 9 and 26 ± 7%, respectively, and the mRNA expression of TNFα, <em>IL</em>-1β, JNK-1, TLR-2, TLR-4, and SOCS-3 in mononuclear cells by 31 ± 12, 22 ± 10, <em>20</em> ± 11, 22 ± 9, 16 ± 7, and 31 ± 10%, respectively (P < 0.05 for all) after 12 wk of exenatide. After a single injection of exenatide, there was a reduction by <em>20</em> ± 7% in free fatty acids, 19 ± 7% in reactive oxygen species generation, 39 ± 11% in nuclear factor-κB binding, 18 ± 9% in TNFα expression, 26 ± 7% in <em>IL</em>-1β expression, 18 ± 7% in JNK-1 expression, 24 ± 12% in TLR-4 expression, and 23 ± 11% in SOCS-3 expression (P < 0.05 for all). The plasma concentrations of monocyte chemoattractant protein-1, matrix metalloproteinase-9, serum amyloid A, and <em>IL</em>-6 were suppressed after 12 wk exenatide treatment by 15 ± 7, <em>20</em> ± 11, 16 ± 7, and 22 ± 12%, respectively (P < 0.05 for all).

CONCLUSIONS

Exenatide exerts a rapid antiinflammatory effect at the cellular and molecular level. This may contribute to a potentially beneficial antiatherogenic effect. This effect was independent of weight loss.

Hepatobiliary transporters are down-regulated in toxic and cholestatic liver injury. Cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (<em>IL</em>-1 beta) are attributed to mediate this regulation, but their particular contribution in vivo is still unknown. Thus, we studied the molecular mechanisms by which Ntcp, Oatp1, Oatp2, and Mrp2 are regulated by proinflammatory cytokines during liver injury. Rats were injected intraperitoneally with either carbon tetrachloride or endotoxin. Inactivation of TNF-alpha and <em>IL</em>-1 beta was achieved by repetitive intraperitoneal injection of etanercept and anakinra, respectively. Messenger RNA (mRNA) levels of transporters and binding activities as well as nuclear protein levels of Ntcp, Oatp2, and Mrp2 transactivators were determined <em>20</em> to 24 hours later. In contrast to <em>IL</em>-1 beta, TNF-alpha inactivation alone fully prevented down-regulation of Ntcp, Oatp1, and Oatp2 mRNA as well as reduced binding activity of hepatocyte nuclear factor 1 (HNF-1) in CCl(4)-induced toxic injury. In endotoxemia, down-regulation of Mrp2, and partially in case of Ntcp, could be prevented by <em>IL</em>-1 beta but not TNF-alpha blockade. However, inactivation of either cytokine led to preservation of HNF1 and partially of retinoid X receptor/retinoic acid receptor (RXR/RAR) binding activity. No effect of anticytokines was seen on pregnane X receptor (PXR) and constitutive androstane receptor (CAR) binding activity as well as nuclear protein mass. In conclusion, TNF-alpha represents the master cytokine responsible for HNF1-dependent down-regulation of Ntcp, Oatp1, and Oatp2 in CCl(4)-induced toxic liver injury. <em>IL</em>-1 beta predominates in a complex signaling network of Ntcp and Mrp2 regulation in cholestatic liver injury. In contrast to in vitro studies, HNF1 and RXR/RAR-independent mechanisms appear to be more important in regulation of Mrp2 and Ntcp gene expression in endotoxemia.

OBJECTIVE

Leflunomide and methotrexate have proven to be efficacious in reducing joint inflammation and slowing destruction in clinical trials of patients with rheumatoid arthritis (RA). This study was conducted to provide more insight into the mechanism of action of these agents in synovial tissue.

METHODS

In a 2-center, prospective, randomized, double-blind clinical trial, we compared leflunomide (<em>20</em> mg/day, after a 3-day 100 mg/day loading dose) and methotrexate (increased stepwise to 15 mg/week) treatment in patients with active RA. Paired synovial tissue biopsy samples were obtained by knee arthroscopy at baseline and after 4 months of treatment. Frozen synovial tissue sections were stained for macrophages (CD68), T cells (CD3), adhesion molecules (intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1]), cytokines (tumor necrosis factor alpha, interleukin-1beta [<em>IL</em>-1beta]), matrix metalloproteinase 1 (MMP-1), and tissue inhibitor of metalloproteinases 1 (TIMP-1).

RESULTS

Paired synovial tissue sections were available in 35 patients (16 taking leflunomide, 19 taking methotrexate). Both drugs displayed equal clinical efficacy, with 8 leflunomide-treated patients (50%) and 10 methotrexate-treated patients (53%) fulfilling the American College of Rheumatology <em>20</em>% response criteria. Both compounds showed similar effects on synovial tissue: reduced numbers of macrophages and reduced ICAM-1 and VCAM-1 expression were noted after 4 months of treatment. Both leflunomide- and methotrexate-treated patients exhibited a decreased MMP-1:TIMP-1 ratio in the synovial tissue. In the subset of patients fulfilling the <em>20</em>% response criteria of the American College of Rheumatology, a more pronounced reduction in the expression of ICAM-1, VCAM-1, <em>IL</em>-1beta, and MMP-1 was found compared with the nonresponders.

CONCLUSIONS

Leflunomide and methotrexate are clinically efficacious drugs that interfere with mechanisms involved in joint inflammation and destruction of joint integrity.

BACKGROUND

Chronic renal failure (CRF) is associated with an increased risk of ischaemic heart disease (IHD), but the mechanisms responsible are controversial. We investigated the relationship of two sets of candidate mechanisms-indices of LDL oxidation and markers of inflammatory activity-with vascular endothelial dysfunction (VED).

METHODS

We carried out cross-sectional analysis of 23 dialysed and 16 non-dialysed CRF patients, 28 healthy controls, and <em>20</em> patients with stable angina and normal renal function. The following were determined: (i) LDL oxidation by Cu(2+) and ultraviolet light, serum autoantibodies to oxidized LDL (oxLDL); (ii) forearm flow-mediated vasodilatation, plasma concentrations of adhesion molecules, and von Willebrand factor (vWF); and (iii) circulating levels of TNF-alpha and <em>IL</em>-6, C-reactive protein (CRP), and fibrinogen.

RESULTS

Endothelium-dependent vasodilatation (EDV) was lower in angina, pre-dialysis, and dialysis CRF patients than in controls (all P<0.005). Compared with controls, vWf (P<0.005) and adhesion molecules (vCAM-1, P<0.005; iCAM-1, P=0.01; E-selectin, P=0.05) were raised in dialysis, and vCAM-1 (P=0.01) in pre-dialysis CRF patients. Dialysed patients had lower HDL cholesterol (P=0.01) and higher triglyceride (P=0.05) than controls, but LDL-oxidation was similar in all groups. Autoantibodies to oxLDL were raised in angina (P<0.005) and pre-dialysis (P=0.006), but were absent in most dialysed patients. Concentrations of IL-6, TNF-alpha, CRP and fibrinogen were elevated in CRF compared with control and angina patients (P<0.005). In the whole population, IL-6 and TNF-alpha correlated negatively with EDV, HDL cholesterol, and positively with triglyceride, blood pressure, vWf, iCAM-1, vCAM-1 and E-selectin (r=-0.43 to +0.70, all P<0.05).

CONCLUSIONS

Endothelial dysfunction is unrelated to LDL oxidation, suggesting that LDL oxidation might not be a major cause of VED in CRF. In contrast VED was more severe in CRF than in angina patients and is associated with increased acute-phase proteins and plasma cytokines, demonstrating a chronic inflammatory state. These observations may explain the VED and increased IHD risk of patients with CRF.

A new quantitative cytometric technique, termed the ArrayScanTM, is described and used to measure NF-kappaB nuclear translocation induced by interleukin (<em>IL</em>)-1 and tumor necrosis factor-alpha (TNFalpha). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappaB was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4-5 min slower than that of Ikappa Balpha degradation. <em>IL</em>-1 receptor antagonist and anti-TypeI <em>IL</em>-1 receptor antiserum on the one hand and anti-TNFalpha and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit <em>IL</em>-1 and TNFalpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFalpha stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent Ikappa Balpha degradation and subsequent NF-kappaB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 microM, while clastolactacystin beta-lactone was a complete inhibitor with an IC50 of 10 microM. The nonselective kinase inhibitor K252a completely inhibited both <em>IL</em>-1 and TNFalpha stimulation in both cell types with an IC50 of 0.4 microM. This concentration, determined after a <em>20</em>-min stimulation, was shown to be comparable with that obtained for inhibition of <em>IL</em>-6 production induced by a 100-fold lower <em>IL</em>-1 and TNFalpha concentration measured after 17 h of stimulation. These results suggest that the ArrayScanTM technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-kappaB.

The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)-mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (<em>IL</em>-3), <em>IL</em>-6, and SCF in the first 5 animals, <em>IL</em>-3/<em>IL</em>-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or <em>IL</em>-3/<em>IL</em>-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of <em>IL</em>-3/<em>IL</em>-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of <em>IL</em>-3/<em>IL</em>-6/SCF, with levels of 10% to <em>20</em>% confirmed by Southern blotting from the nonexpanded <em>IL</em>-3/<em>IL</em>-6/SCF/FLT/STR graft compared with 0.01% in the original <em>IL</em>-3/<em>IL</em>-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.

STAT5, a member of the signal transducers and activators of transcription (STATs), is important in modulating T cell functions through interleukin-2 (<em>IL</em>-2) receptors. Like other STAT proteins, STAT5 undergoes a rapid activation and inactivation cycle upon cytokine stimulation. Tyrosine phosphorylation and dephosphorylation are critical in regulating STAT5 activity. A number of protein tyrosine kinases have been shown to phosphorylate STAT5; however, the phosphatases responsible for STAT5 dephosphorylation remain unidentified. Using CTLL-<em>20</em> as a model system, we provide evidence that tyrosine dephosphorylation of STAT5 subsequent to <em>IL</em>-2-induced phosphorylation occurs in the absence of STAT5 nuclear translocation and new protein synthesis. Nevertheless, down-regulation of the upstream Janus kinase activity during the deactivation cycle of <em>IL</em>-2-induced signaling does involve new protein synthesis. These findings point to the constitutive presence of STAT5 tyrosine phosphatase activity in the cytosolic compartment. We further demonstrate that SHP-2, but not SHP-1, directly dephosphorylates STAT5 in an in vitro tyrosine phosphatase assay with purified proteins. Furthermore, tyrosine-phosphorylated STAT5 associates with the substrate-trapping mutant (Cys ->> Ser) of SHP-2 but not SHP-1. These results suggest a potential role for cytoplasmic protein-tyrosine phosphatases in directly dephosphorylating STAT proteins and in maintaining a basal steady state level of STAT activity.

To investigate the consequences of the impaired parasite-specific immune response in lymphatic filariasis, the effect of concurrent Wuchereria bancrofti infection on the immune response to tetanus toxoid (TT) following tetanus vaccination was studied in <em>20</em> asymptomatic microfilaremic (MF) patients, <em>20</em> patients with chronic lymphatic obstruction/elephantiasis (chronic pathology [CP]), and 10 endemic normal (EN) control individuals at baseline and at 3 and 6 months after TT vaccination. Peripheral blood mononuclear cell (PBMC) proliferative responses to TT before vaccination were not significantly different between the EN control and CP groups, but the MF group showed significantly lower baseline proliferative responses to TT compared with either the EN or CP group. Six months following vaccination, the change in proliferative response to TT was significantly greater in the EN and CP groups than in the MF group. This difference in proliferative response was reiterated in the gamma interferon (IFN-gamma) response in the EN group, in that they increased IFN-gamma production by 400% at 6 months, in contrast to that seen in the filaria-infected groups. In contrast to the IFN-gamma responses, PBMCs from the MF group produced significantly increased levels of TT-specific <em>IL</em>-10 compared with PBMCs from the EN group. Although there was significantly greater TT-specific immunoglobulin G (IgG) production at baseline between the EN and MF groups, postvaccination IgG (and IgG1 isotype) responses did not differ among the groups, whereas TT-specific IgG2, IgG3, and IgG4 were all increased in the EN group compared with the filaria-infected groups. These studies indicate that concurrent infection with W. bancrofti can diminish the immune response to an unrelated antigen by a mechanism that is likely to involve <em>IL</em>-10.

OBJECTIVE

To investigate the difference in acute phase protein responses between patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and spondyloarthropathies (SpA).

METHODS

Circulating levels of cytokines inducing the production of acute phase proteins such as interleukin (<em>IL</em>)-6, <em>IL</em>-1 beta, and tumor necrosis factor (TNF)-alpha, and of cytokine inhibitors such as TNF soluble receptors (TNF-sR55 and TNF-sR75) and <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra), were measured in 2 cohorts of patients. The first cohort included 52 patients with SLE and 22 with RA, and the second included 21 with SLE, <em>20</em> with RA, and 18 with SpA. An examination at the time of blood collection and the Systemic Lupus Activity Measure (SLAM) index were used to assess disease activity in patients with SLE. Serum levels of <em>IL</em>-6 were measured using a biological assay, and concentrations of <em>IL</em>-1 beta, TNF-alpha, TNF-sR55, TNF-sR75, and <em>IL</em>-1ra were assessed by immunoassays.

RESULTS

Although C-reactive protein (CRP) levels were significantly lower in SLE than in RA or SpA, the concentrations of circulating IL-6 or TNF-alpha were higher in SLE. The most striking observation was that TNF-sR levels were significantly higher in SLE than in RA or SpA. The TNF-alpha: TNF-sR ratio was also significantly lower in SLE than in RA. TNF-sR55 and TNF-sR75 levels correlated with disease activity in SLE.

CONCLUSIONS

The weak acute phase protein response in SLE may be explained by a decreased ratio between inducing cytokines and their inhibitors. In addition, TNF-sR may prove a useful biological marker for the followup of SLE, where acute phase protein response is generally low during disease exacerbations.

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (<em>IL</em>-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and <em>IL</em>-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while <em>IL</em>-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked <em>20</em> amino acids in a region of sequence homology with the <em>IL</em>-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.

OBJECTIVE

To investigate the long-term associations of magnesium intake with incidence of diabetes, systemic inflammation, and insulin resistance among young American adults.

METHODS

A total of 4,497 Americans, aged 18-30 years, who had no diabetes at baseline, were prospectively examined for incident diabetes based on quintiles of magnesium intake. We also investigated the associations between magnesium intake and inflammatory markers, i.e., high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and fibrinogen, and the homeostasis model assessment of insulin resistance (HOMA-IR).

RESULTS

During the 20-year follow-up, 330 incident cases of diabetes were identified. Magnesium intake was inversely associated with incidence of diabetes after adjustment for potential confounders. The multivariable-adjusted hazard ratio of diabetes for participants in the highest quintile of magnesium intake was 0.53 (95% CI, 0.32-0.86; P(trend) < 0.01) compared with those in the lowest quintile. Consistently, magnesium intake was significantly inversely associated with hs-CRP, IL-6, fibrinogen, and HOMA-IR, and serum magnesium levels were inversely correlated with hs-CRP and HOMA-IR.

CONCLUSIONS

Magnesium intake was inversely longitudinally associated with incidence of diabetes in young American adults. This inverse association may be explained, at least in part, by the inverse correlations of magnesium intake with systemic inflammation and insulin resistance.

BACKGROUND

Chronic kidney disease (CKD) predisposes to a 10- to <em>20</em>-fold increased cardiovascular risk. Patients undergo accelerated atherogenesis and vascular ageing. We investigated whether telomere attrition, a marker of cell senescence, contributes to this increased mortality risk.

METHODS

This is a cross-sectional study in prevalent haemodialysis patients [n = 175; 98 Males; median (range) age: 66 (23-86) years]. Biochemical markers of oxidative stress and inflammatory status were measured in relation to the patient's leucocyte telomere length. Overall mortality was assessed after a median of 31 (range 2-42) months.

RESULTS

Telomere length was shorter in CKD men, despite women being older (average +/- SD 6.41 +/- 1.23 vs. 6.96 +/- 1.48 kb, P = 0.002). Telomere length was associated with age (rho = -0.18, P = 0.01), fetuin-A (rho = 0.26, P = 0.0004), high-sensitivity C-reactive protein (rho = -0.21, P = 0.005) and IL-6 (rho = -0.17, P = 0.02). In a multivariate logistic regression (pseudo r(2) = 0.14), telomere length was associated with age >65 years (odds ratio: 2.11; 95% CI: 1.10, 4.06), sex (2.01; 1.05, 3.86), fetuin-A (1.85; 0.97, 3.50) and white blood cell count (2.04; 1.02, 4.09). Receiver operating characteristic curves identified a telomere length < 6.28 kb as a fair predictor of mortality. Finally, reduced telomere length was associated with increased mortality, independently of age, gender and inflammation (likelihood ratio 41.6, P < 0.0001), but dependently on fetuin-A levels.

CONCLUSIONS

Age and male gender seem to be important contributors to reduced telomere length in CKD patients, possibly via persistent inflammation. Reduced telomere length also contributes to the mortality risk of these patients through pathways that could involve circulating levels of fetuin-A.

These studies have examined the binding of the three <em>IL</em>-1 ligands, <em>IL</em>-1 alpha, <em>IL</em>-1 beta, and <em>IL</em>-1 receptor antagonist (<em>IL</em>-1 ra), to soluble forms of types I and II <em>IL</em>-1Rs (s<em>IL</em>-1RI and s<em>IL</em>-1RII). This interaction was measured in direct binding experiments, in which the ligands bound to immobilized s<em>IL</em>-1R, and in inhibition experiments, in which s<em>IL</em>-1R in solution inhibited the binding of <em>IL</em>-1 ligands to immobilized s<em>IL</em>-1R. In addition, the effects of s<em>IL</em>-1R on the detection of <em>IL</em>-1 ligands by ELISA were examined. Finally, levels of s<em>IL</em>-1R in synovial fluid samples were determined, and their effects on measurement of <em>IL</em>-1 in these samples were estimated. <em>IL</em>-1 beta bound more avidly to s<em>IL</em>-1RII than <em>IL</em>-1 alpha or <em>IL</em>-1ra, primarily because of a slow dissociation rate. In contrast, <em>IL</em>-1 ra bound more avidly than <em>IL</em>-1 alpha or <em>IL</em>-1 beta to s<em>IL</em>-1RI, again because of a slow dissociation rate. s<em>IL</em>-1RII and s<em>IL</em>-1RI inhibited the detection of <em>IL</em>-1 beta and <em>IL</em>-1ra, respectively, by ELISA. Low levels of s<em>IL</em>-1RI (approximately 1.0-2.5 ng/ml) were present in all synovial fluids, irrespective of the degree of inflammation, and were correlated inversely with the levels of measured <em>IL</em>-1ra. In contrast, higher levels of s<em>IL</em>-1RII (approximately 10-<em>20</em> ng/ml) were found in inflammatory synovial fluids and were not correlated with <em>IL</em>-1ra levels. <em>IL</em>-1 beta could not be detected in any synovial fluid. These results suggest that some <em>IL</em>-1 beta and <em>IL</em>-1ra may be bound in vivo to s<em>IL</em>-1RII and s<em>IL</em>-1RI, respectively, leading to underestimations of cytokine concentrations in body fluids when measured by ELISA.