BACKGROUND

Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions.

OBJECTIVE

The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut.

METHODS

This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment.

RESULTS

Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (<em>20</em> g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and <em>IL</em>-10 levels after immunotherapy in only the active group.

CONCLUSIONS

Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.

BACKGROUND

Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of disorders with, for the most part, an unknown immunopathogenesis. Although onset and disease course differ, the subtypes of JIA share the occurrence of chronic inflammation of the joints, with infiltrations of immunocompetent cells that secrete inflammatory mediators.

OBJECTIVE

To identify a panel of cytokines specifically related to the inflammatory process in JIA.

METHODS

Using a new technology, the multiplex immunoassay, 30 cytokines were measured in plasma of 65 patients with JIA, of which 34 were paired with synovial fluid. These data were compared with plasma of <em>20</em> healthy controls and 9 patients with type I diabetes, a chronic inflammatory disease.

RESULTS

Patients with JIA had, irrespective of their subclassification, significantly higher levels of tumour necrosis factor alpha, macrophage inhibitory factor (MIF), CCL2, CCL3, CCL11, CCL22 and CXCL9 in plasma than controls. In paired plasma and synovial fluid samples of patients with JIA, significantly higher levels of interleukin (IL)6, IL15, CCL2, CCL3, CXCL8, CXCL9 and CXCL10 were present in synovial fluid. Cluster analysis in all patients with JIA revealed a predominant pro-inflammatory cytokine cluster during active disease and a regulatory/anti-inflammatory-related cytokine cluster during remission. Whether a discrimination profile of various cytokines could help in the determination of disease classification was tested.

CONCLUSIONS

It is suggested that several cytokines (IL18, MIF, CCL2, CCL3, CCL11, CXCL9 and CXCL10) may correspond to the activation status during inflammation in JIA and could be instrumental in monitoring disease activity and outcomes of (new) immunotherapies.

<em>IL</em>-12, a heterodimeric cytokine, consists of two disulfide-linked subunits, p40 and p35. We investigated the role of p40 in ligand binding and signal transduction by expressing this subunit alone in COS cells. Culture media of the transfected COS cells exhibited specific dose-dependent binding to KIT225/K6 cells, a human T cell line that expresses <em>IL</em>-12R. Analysis of the culture media by SDS-PAGE and Western blotting demonstrated the presence of 40-kDa monomers and 80-kDa disulfide-linked homodimers. The two p40 species were purified and identified by N-terminal sequencing and proteolytic peptide mapping. Characterization of the p40 proteins for binding and bioactivity showed that both the p40 monomer and dimer inhibited 125I-labeled <em>IL</em>-12 binding to <em>IL</em>-12R, but the 80-kDa species, having a 50% inhibitory concentration (IC50) of <em>20</em> to 70 ng/ml, was at least <em>20</em>-fold more effective than the monomer. Although neither the monomer nor the dimer stimulated human PHA-blast proliferation, the 80-kDa dimer inhibited <em>IL</em>-12-induced proliferation in a dose-dependent manner with an IC50 of 65 ng/ml. The results suggest that the <em>IL</em>-12 p40 subunit contains the essential epitopes for receptor binding. However, a proper conformation required for high affinity binding is achieved only when p40 is associated with a p35 subunit or another p40 subunit. When p40 is associated with a p35 subunit, the heterodimer acts as an agonist mediating biologic activity. However, when p40 associates with another p40, the homodimer behaves as an antagonist in vitro.

Murine <em>IL</em>-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human <em>IL</em>-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of <em>IL</em>-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to <em>20</em>-fold higher levels of <em>IL</em>-10. In addition, <em>IL</em>-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of <em>IL</em>-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of <em>IL</em>-10 production. Kinetic studies indicated that <em>IL</em>-10 was produced relatively late as compared with other cytokines. Maximal <em>IL</em>-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal <em>IL</em>-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of <em>IL</em>-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for <em>IL</em>-10 in later phases of the immune response.

Demonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-alpha and interleukin (<em>IL</em>)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-alpha, <em>IL</em>-6, and <em>IL</em>-8 with an IC(50) = 170 to 3<em>20</em> nM. Prophylactic administration of TPCA-1 at 3, 10, or <em>20</em> mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of <em>IL</em>-1beta, <em>IL</em>-6, TNF-alpha, and interferon-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at <em>20</em> mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.

The human T cell receptor for antigen (Ti) has recently been identified on <em>IL</em>-2 dependent T cell clones as a 90 kd disulfide-linked heterodimer comprised of one 49-51 kd alpha (alpha) and one 43 kd beta (beta) chain. These subunits are noncovalently associated with a monomorphic <em>20</em>-25 kd T3 molecule. Here, we produce monoclonal antibodies to a human tumor (REX) derived from an earlier stage of thymic differentiation in order to determine whether clonotypic structures are expressed and to define the ontogeny of Ti. The results of SDS-PAGE and peptide map analyses indicate that an homologous T3-associated heterodimer is synthesized and expressed by REX. This glycoprotein shares several peptides in common with clonotypic structures on an <em>IL</em>-2 dependent T cell clone. In addition, similar Ti related molecules appear during intrathymic ontogeny in parallel with surface T3 expression. The latter findings provide the structural basis for the immunological competence observed exclusively within the T3+ thymocyte compartment.

BACKGROUND

Cigarette smoke mediated oxidative stress and inflammatory events in the airway and alveolar epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Previously, individual cell lines were used to assess the oxidative and proinflammatory effects of cigarette smoke with confounding results. In this study, a panel of human and rodent transformed epithelial cell lines were used to determine the effects of cigarette smoke extract (CSE) on oxidative stress markers, cell toxicity and proinflammatory cytokine release and compared the effects with that of primary human small airway epithelial cells (SAEC).

METHODS

Primary human SAEC, transformed human (A549, H1299, H441), and rodent (murine MLE-15, rat L2) alveolar epithelial cells were treated with different concentrations of CSE (0.2-10%) ranging from <em>20</em> min to 24 hr. Cytotoxicity was assessed by lactate dehydrogenase release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide. Glutathione concentration was measured by enzymatic recycling assay and 4-hydroxy-2-nonenal levels by using lipid peroxidation assay kit. The levels of proinflammatory cytokines (e.g. <em>IL</em>-8 and <em>IL</em>-6) were measured by ELISA. Nuclear translocation of the transcription factor, NF-kappaB was assessed by immunocytochemistry and immunoblotting.

RESULTS

Cigarette smoke extract dose-dependently depleted glutathione concentration, increased 4-hydroxy-2-nonenal (4-HNE) levels, and caused necrosis in the transformed cell lines as well as in SAEC. None of the transformed cell lines showed any significant release of cytokines in response to CSE. CSE, however, induced IL-8 and IL-6 release in primary cell lines in a dose-dependent manner, which was associated with the nuclear translocation of NF-kappaB in SAEC.

CONCLUSIONS

This study suggests that primary, but not transformed, lung epithelial cells are an appropriate model to study the inflammatory mechanisms in response to cigarette smoke.

Studies suggest that stress can be a co-factor for the initiation and progression of cancer. The catecholamine stress hormone, norepinephrine (NE), may influence tumor progression by modulating the expression of factors implicated in angiogenesis and metastasis. The goal of this study was to examine the influence of NE on the expression of VEGF, <em>IL</em>-8, and <em>IL</em>-6 by the human melanoma cell lines, C8161, 1174MEL, and Me18105. Cells were treated with NE and levels of VEGF, <em>IL</em>-8, and <em>IL</em>-6 were measured using ELISA and real-time PCR. The expression of beta-adrenergic receptors (beta-ARs) mRNA and protein were also assessed. Finally, immunohistochemistry was utilized to examine the presence of beta1- and beta2-AR in primary and metastatic human melanoma biopsies. We show that NE treatment upregulated production of VEGF, <em>IL</em>-8, and <em>IL</em>-6 in C8161 cells and to a lesser extent 1174MEL and Me18105 cells. The upregulation was associated with induced gene expression. The effect on C8161 cells was mediated by both beta1- and beta2-ARs. Furthermore, 18 of <em>20</em> melanoma biopsies examined expressed beta2-AR while 14 of <em>20</em> melanoma biopsies expressed beta1-AR. Our data support the hypothesis that NE can stimulate the aggressive potential of melanoma tumor cells, in part, by inducing the production VEGF, <em>IL</em>-8, and <em>IL</em>-6. This line of research further suggests that interventions targeting components of the activated sympathetic-adrenal medullary (SAM) axis, or the utilization of beta-AR blocking agents, may represent new strategies for slowing down the progression of malignant disease and improving cancer patients' quality of life.

In previous studies it has been shown that the bacterial endotoxin LPS induces an initial burst of inflammatory cytokine synthesis in human monocytes, which is followed by substantial <em>IL</em>-10 production; the <em>IL</em>-10 then down-regulates the inflammatory cytokine production as well as <em>IL</em>-10 production itself. Herein we tested the hypothesis that <em>IL</em>-10 production in human monocytes is under control of one of the cytokines induced by LPS. Accordingly, we cocultured purified human peripheral blood monocytes with a panel of cytokines including TNF-alpha, <em>IL</em>-1 alpha, <em>IL</em>-1 beta, <em>IL</em>-6, granulocyte macrophage-CSF, transforming growth factor-beta, and IFN-alpha and then measured <em>IL</em>-10 mRNA production using a semiquantitative reverse transcription-polymerase chain reaction technique. We found that TNF-alpha had a major effect on <em>IL</em>-10 mRNA production, inducing a <em>20</em>- to 1<em>20</em>-fold increase over baseline production. In contrast, <em>IL</em>-1 alpha, <em>IL</em>-1 beta, <em>IL</em>-6, granulocyte macrophage-CSF, transforming growth factor-beta, and IFN-alpha had little effect (< 3-fold). The induction of <em>IL</em>-10 mRNA by TNF-alpha in monocytes was dose dependent and began between 8 and 24 h after the addition of TNF-alpha; this suggests that the increased <em>IL</em>-10 mRNA level was due to de novo mRNA synthesis rather than mRNA stabilization; this latter finding was corroborated by actinomycin-D time course studies, which showed that the half-life of <em>IL</em>-10 was less than 1 h and was not significantly altered by TNF-alpha. These studies concerning <em>IL</em>-10 mRNA induction by TNF-alpha were corroborated by studies of <em>IL</em>-10 protein secretion: TNF-alpha alone, but not <em>IL</em>-1 alpha, <em>IL</em>-1 beta, or <em>IL</em>-6 induces substantial <em>IL</em>-10 secretion. Furthermore, LPS induces a large amount of <em>IL</em>-10 secretion that is largely inhibited (50 to 75%) by anti-TNF-alpha but not by antibodies to other inflammatory cytokines. Finally, TNF-alpha augments LPS-induced <em>IL</em>-10 secretion. Taken together, these findings suggest that TNF-alpha is unique among the inflammatory cytokines in its role as an inducer of <em>IL</em>-10 in human monocytes, as such, it induces a molecule that provides negative feedback to its own production.

BACKGROUND

The immune deficiency of human immunodeficiency virus (HIV) infection is not fully corrected with ARV therapy. Interleukin-7 (IL-7) can boost CD4 T-cell counts, but optimal dosing and mechanisms of cellular increases need to be defined.

METHODS

We performed a randomized placebo-controlled dose escalation (10, 20 and 30 µg/kg) trial of 3 weekly doses of recombinant human IL-7 (rhIL-7) in ARV-treated HIV-infected persons with CD4 T-cell counts between 101 and 400 cells/µL and plasma HIV levels <50 copies/mL. Toxicity, activity and the impact of rhIL-7 on immune reconstitution were monitored.

RESULTS

Doses of rhIL-7 up to 20 µg/kg were well tolerated. CD4 increases of predominantly naive and central memory T cells were brisk (averaging 323 cells/µL at 12 weeks) and durable (up to 1 year). Increased cell cycling and transient increased bcl-2 expression were noted. Expanded cells did not have the characteristics of regulatory or activated T cells. Transient low-level HIV viremia was seen in 6 of 26 treated patients; modest increases in total levels of intracellular HIV DNA were proportional to CD4 T-cell expansions. IL-7 seemed to increase thymic output and tended to improve the T-cell receptor (TCR) repertoire in persons with low TCR diversity.

CONCLUSIONS

Three weekly doses of rhIL-7 at 20 µg/kg are well tolerated and lead to a dose-dependent CD4 T-cell increase and the broadening of TCR diversity in some subjects. These data suggest that this rhIL-7 dose could be advanced in future rhIL-7 clinical studies.

BACKGROUND

NCT0047732.

OBJECTIVE

The purpose of this study was to determine the relative contributions of individual proinflammatory cytokines and chemokines to the triggering of preterm labor.

METHODS

Eighteen chronically instrumented pregnant rhesus monkeys at 135 +/- 3 days gestation (term = 167 days) received 1 of 5 intraamniotic infusions: (1) interleukin-1beta (<em>IL</em>-1beta) (10 microg; n = 5), (2) tumor necrosis factor-alpha (TNF-alpha) (10-100 microg; n = 5), (3) <em>IL</em>-6 (<em>20</em> microg twice a day; n = 2), (4) <em>IL</em>-8 (<em>20</em> microg twice a day; n = 2), and (5) saline control (n = 4). Primary study outcomes were the mean uterine hourly contraction area (mm Hg x s/h) in 24 hours during peak response to cytokine infusion (all groups) and the interval from cytokine infusion until labor onset (<em>IL</em>-1beta, <em>IL</em>-6, and <em>IL</em>-8 groups). Secondary outcomes were quantities of amniotic fluid cytokines and chemokines (<em>IL</em>-1beta, TNF-alpha, <em>IL</em>-6, and <em>IL</em>-8), prostaglandins E2 and F2alpha, leukocytes, and matrix metalloproteinase-9 (MMP-9). Histopathology of fetal lungs and placental membranes was assessed.

RESULTS

IL-1beta stimulated the most intense contraction patterns, resulting in preterm labor in all cases. TNF-alpha induced a variable degree of uterine activity among individual animals stimulating either preterm labor (n = 2) or a uterine contraction pattern of moderate intensity (n = 3). Despite prolonged elevations in amniotic fluid levels, neither IL-6 nor IL-8 induced preterm labor or an increase in uterine activity until near term. The mean interval from the initiation of IL-6 and IL-8 infusion to the onset of labor was significantly longer than after IL-1beta (21.9 vs 1.1 days; P < .01), and did not differ from the saline control group (27.6 days; P = NS). Intraamniotic infusion of IL-1beta or TNF-alpha was associated with significant elevations in all tested amniotic fluid cytokines, IL-8, prostaglandins, MMP-9 and leukocytes compared with gestational age-matched saline controls. IL-6 and IL-8 infusions were not associated with increases in IL-1beta or TNF-alpha and only produced a moderate increase in amniotic fluid prostaglandins. All cytokine infusions induced histologic chorioamnionitis and an accumulation of neutrophils in fetal lungs.

CONCLUSIONS

Preterm labor was induced by intraamniotic infusions of IL-1beta and TNF-alpha, but not by IL-6 or IL-8 although inflammatory changes in fetal membranes and lungs were uniformly present. Our results indicate a primary role for IL-1beta and TNF-alpha in the triggering of preterm labor associated with inflammation or infection.

Severe acute respiratory syndrome (SARS) presented as an atypical pneumonia that progressed to acute respiratory distress syndrome in approximately <em>20</em>% of cases and was associated with a mortality of about 10%. The etiological agent was a novel coronavirus (CoV). Angiotensin-converting enzyme 2 is the functional receptor for SARS-CoV; DC-SIGN and CD<em>20</em>9L (L-SIGN) can enhance viral entry. Although the virus infects the lungs, gastrointestinal tract, liver, and kidneys, the disease is limited to the lungs, where diffuse alveolar damage is accompanied by a disproportionately sparse inflammatory infiltrate. Pro-inflammatory cytokines and chemokines, particularly IP-10, <em>IL</em>-8, and MCP-1, are elevated in the lungs and peripheral blood, but there is an unusual lack of an antiviral interferon (IFN) response. The virus is susceptible to exogenous type I IFN but suppresses the induction of IFN. Innate immunity is important for viral clearance in the mouse model. Virus-specific neutralizing antibodies that develop during convalescence prevent reinfection in animal models.

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (<em>IL</em>-17), C-C chemokine ligand <em>20</em> (CCL<em>20</em>)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/<em>IL</em>-8, and lower interferon-gamma and <em>IL</em>-22 levels than in control cultures. Exogenous PGE(2) and <em>IL</em>-23 synergized in inducing <em>IL</em>-17, whereas indomethacin and <em>IL</em>-23 blockade drastically reduced <em>IL</em>-17 but not interferon-gamma production. Furthermore, <em>IL</em>-1 but not tumor necrosis factor was absolutely required for <em>IL</em>-17 production. PGE(2) doubled the frequency of CD4+ T cells producing <em>IL</em>-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of <em>IL</em>-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and <em>IL</em>-1beta. These results identify PGE(2) and <em>IL</em>-23 as participating in the expansion of CD4+ T cells endowed with high <em>IL</em>-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.

First described in 1971, adult-onset Still's disease (AOSD) is a rare multisystemic disorder considered as a complex (multigenic) autoinflammatory syndrome. A genetic background would confer susceptibility to the development of autoinflammatory reactions to environmental triggers. Macrophage and neutrophil activation is a hallmark of AOSD which can lead to a reactive hemophagocytic lymphohistiocytosis. As in the latter disease, the cytotoxic function of natural killer cells is decreased in patients with active AOSD. <em>IL</em>-18 and <em>IL</em>-1β, two proinflammatory cytokines processed through the inflammasome machinery, are key factors in the pathogenesis of AOSD; they cause <em>IL</em>-6 and Th1 cytokine secretion as well as NK cell dysregulation leading to macrophage activation. The clinico-biological picture of AOSD usually includes high spiking fever with joint symptoms, evanescent skin rash, sore throat, striking neutrophilic leukocytosis, hyperferritinemia with collapsed glycosylated ferritin ((<em>20</em>%), and abnormal liver function tests. According to the clinical presentation of the disease at diagnosis, two AOSD phenotypes may be distinguished: i) a highly symptomatic, systemic and feverish one, which would evolve into a systemic (mono- or polycyclic) pattern; ii) a more indolent one with arthritis in the foreground and poor systemic symptomatology, which would evolve into a chronic articular pattern. Steroid- and methotrexate-refractory AOSD cases benefit now from recent insights into autoinflammatory disorders: anakinra seems to be an efficient, well tolerated, steroid-sparing treatment in systemic patterns; tocilizumab seems efficient in AOSD with active arthritis and systemic symptoms while TNFα-blockers could be interesting in chronic polyarticular refractory AOSD.

OBJECTIVE

Bacterial proteoglycan-derived muramyl dipeptide (MDP) activates the intracellular NOD2/CARD15 gene product. How intestinal epithelial cells take up MDP is poorly understood. We hypothesized that the intestinal apical di-/tripeptide transporter, hPepT1, transports MDP, thereby activating downstream pathways similar to nuclear factor kappa B (NF-kappaB).

METHODS

Time- and concentration-dependent (3)H-MDP uptakes were studied in Caco2/bbe (C2) cell monolayers where hPepT1 expression was either over- or underexpressed, using an inducible adenovirus system or silencing RNA (siRNA), respectively. NF-kappaB activation and interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) release were determined by enzyme-linked immunosorbent assay. NOD2/CARD15 expression was inhibited by siRNA. MDP in human duodenal, cecal, and stool samples was measured.

RESULTS

MDP, but not its isoforms, inhibited uptake of glycosylsarcosine in C2 cells, indicating stereoselective and competitive inhibition. Approximately 90% of the MDP was cytosolic, showing uptake rather than binding. The K m for MDP uptake was 4.3 mmol/L. Cells overexpressing hPepT1 showed increased Gly-Sar and MDP uptake, whereas decreased uptake was observed after hPepT1 siRNA-inhibition. MDP treatment activated NF-kappaB, resulting in IL-8 release, an effect blocked by siRNA-inhibited expression of NOD2/CARD15. MDP content in cecal and stool samples (in normal subjects) was 20-87 micromol/L, but undetectable in duodenal fluid.

CONCLUSIONS

In colonic epithelial cells, MDP is taken up by hPepT1 and activates NF-kappaB and chemokine production. Because hPepT1 expression in chronic colonic inflammation is increased, this may play an important role in promoting colonocyte participation in host defense and pathogen clearance through increased uptake of MDP.

OBJECTIVE

To examine the role of cytokine interleukin-1beta (IL-1beta) in retinal capillary cell death in diabetes.

METHODS

The effect of glucose on the expression of IL-1beta was measured in the bovine retinal endothelial cells. The role of IL-1beta in the accelerated endothelial cell loss was determined by investigating the effect of human recombinant IL-1beta on their apoptosis in normal and high glucose conditions, and was confirmed using interleukin-1 receptor antagonist (IL-1ra).

RESULTS

High glucose increased IL-1beta expression by 60% compared with cells incubated in 5 mM glucose (p<0.05). Incubation of cells with IL-1beta increased NO levels by about 80% and activated NF-kappaB by 40%. In the same cells apoptosis was increased by 70% and caspase-3 activity was increased by 40%. Supplementation of IL-1beta in 20 mM glucose medium further increased nitric oxide and NF-kappaB, and accelerated apoptosis, and addition of IL-1ra significantly decreased glucose induced abnormalities and apoptosis.

CONCLUSIONS

IL-1beta accelerates apoptosis of retinal capillary cells via activation of NF-kappaB, and the process is exacerbated in high glucose conditions. These studies suggest a possible role of IL-1beta in the development of retinopathy in diabetes, and offer a possible rationale to test IL-1beta receptor antagonists to inhibit the development of diabetic retinopathy.

Bcl-2, a proto-oncogene that can block apoptosis, was found to be expressed throughout the thymic medulla, but in only scattered cells in the thymic cortex. In order to determine the precise distribution of Bcl-2 protein during thymocyte development, we utilized mAb specific for either mouse or human Bcl-2. Thymocyte subpopulations were assessed using three-color flow cytometry and a saponin-permeabilization method. Staining of adult mouse and human thymocytes was comparable, with <em>20</em> to 35% of cells expressing Bcl-2. Bcl-2 was expressed in nearly all CD4+ and CD8+, and CD3hi cells, but in only 5 to 10% of CD4+8+ cells. The CD4-8- population was more variable, with 25 to 40% of human cells and 65 to 80% of murine cells expressing Bcl-2. In sorted adult murine CD4-8- cells, the very immature Pgp-1+/<em>IL</em>-2R alpha- subset had a high percentage of Bcl-2+ cells. Bcl-2 expression was also examined during murine fetal development. At fetal day 15.5 to 16.5, 60 to 70% of total thymocytes expressed Bcl-2. By fetal day 17.5, overall Bcl-2 expression fell to adult levels of <em>20</em> to 30%. Bcl-2 was present in peripheral T cells from lymph node, spleen, and peripheral blood at uniformly high levels. In vitro stimulation with anti-CD3 or anti-TCR antibodies increased Bcl-2 expression in total thymocyte cultures, but could not induce Bcl-2 expression in CD4+8+ cells, even with the addition of a variety of cytokines. These data suggest that early double negative thymocytes express Bcl-2 but lose Bcl-2 with differentiation to the double positive stage. Thymocytes regain Bcl-2 during selection to a single positive state and retain Bcl-2 in the periphery.

This study demonstrates interleukin 6 (<em>IL</em>-6) production and release by human glioblastomas. Twenty glioblastoma cell lines were tested for <em>IL</em>-6 bioactivity using an <em>IL</em>-6-dependent cell line (7TD1). All of the lines tested with one exception (LN-229) constitutively released <em>IL</em>-6. A significant induction of <em>IL</em>-6 production and secretion was observed when LN-229 cells were treated with interleukin 1 beta (<em>IL</em>-1 beta) or tumor necrosis factor alpha. Various amounts of <em>IL</em>-6 mRNA were found in five of six cell lines tested. <em>IL</em>-6 mRNA was detected in line LN-229 only when the cells were treated with <em>IL</em>-1 beta or tumor necrosis factor alpha, confirming the bioassay data. Glioblastoma cells also produce <em>IL</em>-6 in vivo. (a) <em>IL</em>-6 activity was detected in 11 of 13 cerebrospinal fluids and five of five tumor cyst fluids. (b) <em>IL</em>-6 mRNA was found in four of four tumors. (c) Immunohistochemical analysis showed <em>IL</em>-6 within the tumor cells in 15 of <em>20</em> glioblastoma sections. In conclusion, biologically active <em>IL</em>-6 is released by almost all glioblastomas both in vitro and in vivo. The elevated levels of serum acute phase proteins and immune complexes found in glioblastoma patients may be the result of this secretion.

Interleukin 1 (<em>IL</em>-1) and tumor necrosis factor alpha (TNFalpha) are known to induce production of reactive oxygen species (ROS), which have been suggested to act as second messengers. Here we demonstrate that ROS production by bovine chondrocytes upon cytokine stimulation induces c-jun expression. Since c-jun expression is regulated by its own gene product via phosphorylation by c-Jun NH2-terminal kinases (JNKs), we investigated if cytokines and ROS could modulate JNK activity in chondrocyte monolayer cultures. Treatment of bovine chondrocytes with both <em>IL</em>-1 and TNFalpha leads to rapid induction of JNK activity, stimulating JNK activity 7- and <em>20</em>-fold, respectively. Importantly, the observation that antioxidant treatment antagonizes <em>IL</em>-1 and TNFalpha activation of JNK provides strong evidence that ROS can act as mediators of JNK activity. Moreover, potent activation of JNK is also observed by direct addition of the ROS hydrogen peroxide (H2O2) to the chondrocyte cultures. Nitric oxide (NO), a multifunctional ROS, also appears to simulate JNK, albeit to a lesser extent. These findings identify JNK as another molecular target for the actions of NO and H2O2. In addition, the inhibitory effect of diphenyleneiodonium on JNK activation implicates the involvement of flavonoid-containing enzymes in the ROS-mediated signaling process. Overstimulation of JNK activity by excessive production of ROS may, therefore, underlie pathological conditions such as arthritis and cancer.

Hormonal and inflammatory responses to low-intensity resistance exercise with vascular occlusion were studied. Subjects (n = 6) performed bilateral leg extension exercise in the seated position, with the proximal end of their thigh compressed at 214 +/- 7.7 (SE) mmHg throughout the session of exercise by means of a pressure tourniquet. Mean intensity and quantity of the exercise were <em>20</em>% of 1 repetition maximum and 14 repetitions x 5 sets, respectively. In each set, the subjects repeated the movement until exhaustion. Plasma concentrations of growth hormone (GH), norepinephrine (NE), lacate (La), lipid peroxide (LP), interleukin-6 (<em>IL</em>-6), and activity of creatine phosphokinase (CPK) were measured before and after the exercise was finished and the tourniquet was released. Concentrations of GH, NE, and La consistently showed marked, transient increases after the exercise with occlusion, whereas they did not change a great deal after the exercise without occlusion (control) done at the same intensity and quantity. Notably, concentration of GH reached a level approximately 290 times as high as that of the resting level 15 min after the exercise. <em>IL</em>-6 concentration showed a much more gradual increase and was maintained at a slightly higher level than in the control even 24 h after exercise. Concentrations of LP and CPK showed no significant change. The results suggest that extremely light resistance exercise combined with occlusion greatly stimulates the secretion of GH through regional accumulation of metabolites without considerable tissue damage.

The seemingly inexorable decline in insulin independence after islet transplant alone (ITA) has raised concern about its clinical utility. We hypothesized that induction immunosuppression therapy determines durability of insulin independence. We analyzed the proportion of insulin-independent patients following final islet infusion in four groups of ITA recipients according to induction immunotherapy: University of Minnesota recipients given FcR nonbinding anti-CD3 antibody alone or T cell depleting antibodies (TCDAb) and TNF-α inhibition (TNF-α-i) (group 1; n = 29); recipients reported to the Collaborative Islet Transplant Registry (CITR) given TCDAb+TNF-α-i (group 2; n = <em>20</em>); CITR recipients given TCDAb without TNF-α-i (group 3; n = 43); and CITR recipients given <em>IL</em>-2 receptor antibodies (<em>IL</em>-2RAb) alone (group 4; n = 177). Results were compared with outcomes in pancreas transplant alone (PTA) recipients reported to the Scientific Registry of Transplant Recipients (group 5; n = 677). The 5-year insulin independence rates in group 1 (50%) and group 2 (50%) were comparable to outcomes in PTA (group 5: 52%; p>)0.05) but significantly higher than in group 3 (0%; p = 0.001) and group 4 (<em>20</em>%; p = 0.02). Induction immunosuppression was significantly associated with 5-year insulin independence (p = 0.03), regardless of maintenance immunosuppression or other factors. These findings support potential for long-term insulin independence after ITA using potent induction therapy, with anti-CD3 Ab or TCDAb+TNF-α-i.

BACKGROUND

The immune responses in patients with novel A(H1N1) virus infection (nvA(H1N1)) are incompletely characterized. We investigated the profile of Th1 and Th17 mediators and interferon-inducible protein-10 (IP-10) in groups with severe and mild nvA(H1N1) disease and correlated them with clinical aspects.

METHODS

Thirty-two patients hospitalized with confirmed nvA(H1N1) infection were enrolled in the study: 21 patients with nvA(H1N1)-acute respiratory distress syndrome (ARDS) and 11 patients with mild disease. One group of <em>20</em> patients with bacterial sepsis-ARDS and another group of 15 healthy volunteers were added to compare their cytokine levels with pandemic influenza groups. In the nvA(H1N1)-ARDS group, the serum cytokine samples were obtained on admission and 3 days later. The clinical aspects were recorded prospectively.

RESULTS

In the nvA(H1N1)-ARDS group, obesity and lymphocytopenia were more common and IP-10, interleukin (IL)-12, IL-15, tumor necrosis factor (TNF)α, IL-6, IL-8 and IL-9 were significantly increased versus control. When comparing mild with severe nvA(H1N1) groups, IL-6, IL-8, IL-15 and TNFα were significantly higher in the severe group. In nonsurvivors versus survivors, IL-6 and IL-15 were increased on admission and remained higher 3 days later. A positive correlation of IL-6, IL-8 and IL-15 levels with C-reactive protein and with>> 5-day interval between symptom onset and admission, and a negative correlation with the PaO(2):FiO(2) ratio, were found in nvA(H1N1) groups. In obese patients with influenza disease, a significant increased level of IL-8 was found. When comparing viral ARDS with bacterial ARDS, the level of IL-8, IL-17 and TNFα was significantly higher in bacterial ARDS and IL-12 was increased only in viral ARDS.

CONCLUSIONS

In our critically ill patients with novel influenza A(H1N1) virus infection, the hallmarks of the severity of disease were IL-6, IL-15, IL-8 and TNFα. These cytokines, except TNFα, had a positive correlation with the admission delay and C-reactive protein, and a negative correlation with the PaO(2):FiO(2) ratio. Obese patients with nvA(H1N1) disease have a significant level of IL-8. There are significant differences in the level of cytokines when comparing viral ARDS with bacterial ARDS.

Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8 T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated <em>20</em> patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (<em>IL</em>-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December <em>20</em>02 and the last November <em>20</em>03. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January <em>20</em>06. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-gamma or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with <em>IL</em>-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division <em>20</em>-30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38(+) blasts in a manner that increased with consecutive division number. These differentiated CD38(+) B cells divided faster than did CD38(-) memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38(+) and CD38(-) cells present in cultures of memory B cells was increased by <em>IL</em>-10. In contrast, <em>IL</em>-2 enhanced proliferation of CD38(-) and CD38(+) memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.