Immunosuppressive parameters in serum of ovarian cancer patients change during the disease course.

Neoplastic cells can escape immune control leading to cancer growth. Regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) are crucial in immune escape. TAM are divided based on their immune profile, M1 are immunostimulatory while M2 are immunosuppressive. Research so far has mainly focused on the intratumoral behavior of these cells. This study, on the other hand, explored the systemic changes of the key metabolites [IL-4 (interleukin), IL-13, arginase, IL-10, VEGF-A (vascular endothelial growth factor), CCL-2 (chemokine (C-C) motif ligand 2) and TGF-ß (transforming growth factor)] linked to Treg, MDSC and TAM during the course of the disease in ovarian and fallopian tube cancer patients. Serum samples were therefore analyzed at diagnosis, after (interval)-debulking surgery and after chemotherapy (paclitaxel-carboplatin). We also determined galectin-1 (gal-1), involved in angiogenesis and tumor-mediated immune evasion. We found significantly lower levels of IL-10, VEGF-A, TGF-ß and arginase and higher levels of gal-1 after chemotherapy compared to diagnosis. After debulking surgery, a decrease in IL-10 was significant. Gal-1 and CCL-2 appeared independent prognostic factors for progression-free and overall survival (OS) (multivariate analysis). These results will help us in the decision making of future therapies in order to further modulate the immune system in a positive way.

CD28/CTLA-4/B7 costimulatory pathway blockade affects regulatory T-cell function in autoimmunity.

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA-4 on effector T (Teff) cells, its immune-modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA-4Ig at day 7 and 9 after immunization, when myelin-reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL-17 and IFN-γ. Importantly, CTLA-4Ig treatment resulted in a transient reduction of Ki67 and CTLA-4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.

Airway inflammation in patients with chronic non-asthmatic cough.

INTRODUCTION: Chronic non-asthmatic cough (CC) is a clinical challenge and underlying pathophysiological mechanisms remain still not completely understood. One of the most common comorbidities in CC is gastro-oesophageal reflux disease (GORD). Airway epithelium damage can contribute to airway inflammation in CC. AIMS: We studied airway inflammation in patients with CC compared to healthy controls. Patients with GORD were treated with proton pump inhibitors (PPI) and cough response to PPI was evaluated. PATIENTS AND METHODS: Sputum was induced in 41 adults with CC and 20 healthy non-smokers who were age and sex matched. We compared sputum differential cell count by cytospin and cytokine and chemokine production at the mRNA and/or protein levels by real-time (RT)-PCR and cytokine bead array (CBA), between patients with CC and healthy subjects. Furthermore we studied airway inflammation in patients with different comorbidities. RESULTS: No differences in sputum differential cell counts were observed between patients with CC and healthy subjects. Sputum monocyte chemoattractant protein-1 (MCP-1) protein levels were significantly higher in patients when compared to controls. Thymic stromal lymphopoietin (TSLP) mRNA was significantly more often expressed in sputum of patients with CC than from healthy controls. Sputum transforming growth factor (TGF)-ß levels did not differ between patients and controls, but were significantly lower in the PPI responders compared to the non-responders; p=0.047. There is no evidence for impaired T helper cell (Th)1/Th2/Th17 balance in CC. Patients with reflux oesophagitis (RO) have significantly more sputum eosinophils than patients without RO. CONCLUSIONS: CC is a condition presenting with different disease phenotypes. High sputum MCP-1 levels are present in a large group of patients with CC and a majority of these patients with CC have increased sputum TSLP levels, most likely produced by damaged airway epithelial cells.

Pepsin and bile acids in induced sputum of chronic cough patients.

One of the theories which explain, why gastroesophageal reflux disease (GORD) may provoke cough, is the occurrence of aspiration of gastric content into the airways. The aim of the study was to assess the presence of aspiration markers: pepsin and bile acids (BA) in induced sputum in gastroesophageal reflux-related (GOR-related) chronic cough (CC) patients. Forty-one CC patients and 20 healthy controls were enrolled in the study. GORD as cause of CC was diagnosed by presence of GORD-related symptoms, gastroscopy and/or improvement of cough upon treatment with proton pump inhibitors (PPI). Patients were divided into two groups based on the response to PPI treatment. In all patients and healthy controls induced sputum was obtained and differential cell counts were calculated. Levels of pepsin and BA were measured in sputum supernatants. Pepsin was detectable in 48.8% samples in CC patients and in 60% healthy controls (p = NS). In pepsin positive samples no significant difference in pepsin concentration could be found between CC patients and control subjects. Pepsin levels in pepsin positive samples were significantly decreased in patients treated with PPI compared to non-treated patients. BA were detectable in 56% samples of CC patients and in 70% healthy controls (p = NS). BA concentration in BA positive samples in CC group was not different from healthy controls. There was also no difference when comparing patients who took PPI and those who did not. Patients characterized as PPI-responders and PPI-non-responders had similar pepsin and BA concentrations. Airway cellularity was not significantly different between groups of patients with or without pepsin or BA in induced sputum. Our results demonstrated the lack of differences in gastric content aspiration between patients with probable GOR-related CC and healthy control subjects. This might imply that the reflex cough theory may be more relevant than the reflux-associated aspiration theory in the pathophysiology of GORinduced chronic cough.

Elevated expression of both mRNA and protein levels of IL-17A in sputum of stable Cystic Fibrosis patients.

BACKGROUND: T helper 17 (Th17) cells can recruit neutrophils to inflammatory sites through production of IL-17, which induces chemokine release. IL-23 is an important inducer of IL-17 and IL-22 production. Our aim was to study the role of Th17 cells in cystic fibrosis (CF) lung disease by measuring IL-17 protein and mRNA levels and IL-22 and IL-23 mRNA in sputum of clinically stable CF patients and by comparing these levels with healthy controls. METHODS: Sputum induction was performed in adult CF patients outside of an exacerbation and healthy control subjects. IL-17A protein levels were measured in supernatants with cytometric bead array (CBA) and RNA was isolated and quantitative RT-PCR was performed for IL-17A, IL-22 and IL-23. RESULTS: We found significantly higher levels of IL-17A protein and mRNA levels (both: p < 0.0001) and IL-23 mRNA levels (p < 0.0001) in the sputum of CF group as compared to controls. We found very low levels of IL-22 mRNA in the CF group. The levels of IL-17 and IL-23 mRNA were higher in patients chronically infected with Pseudomonas aeruginosa (P. aeruginosa) as compared to those who were not chronically infected with P. aeruginosa. The presence of Staphylococcus aureus (S. aureus) on sputum did not affect the IL-17 or IL-23 levels. There was no correlation between IL-17 or IL-23 levels and FEV1 nor sputum neutrophilia. CONCLUSION: The elevated levels of IL-17 and IL-23 might indicate that Th17 cells are implicated in the persistent neutrophil infiltration in CF lung disease and chronic infection with P. aeruginosa.

Murine bone marrow chimeras developing autoimmunity after CTLA-4-blockade show an expansion of T regulatory cells with an activated cytokine profile.

Autoimmune adverse events are a concern in patients treated with blocking anti-CTLA-4-mAb for solid and hematological tumors. Patient and mouse data on the contribution of a quantitative or qualitative defect of regulatory T cells (T(reg)) in this autoimmune phenomenon are conflicting. We have previously shown that a treatment course with blocking anti-CTLA-4-mAb in murine allogeneic bone marrow chimeras induces an antileukemic response in close association with systemic autoimmunity. Here, we used this model to investigate the effect of CTLA-4-blocking therapy on the kinetics of T(reg) frequency and function. As previously published, CTLA-4-blocking treatment, initiated on day 20 after bone marrow transplantation, led to overt autoimmunity by day 35. CD4(+)Foxp3(+) T(reg) frequency was determined (flowcytometry) on day 21, 23, 25 and 35: treated chimeras showed an expansion of CD4(+)Foxp3(+) T(reg) frequencies on day 25 and 35, without a prior frequency decrease. The T(reg) expansion occurred selectively in the recipient-derived CD4+ T-cell compartment. In vitro, purified CD4(+)CD25(+)FR4(high) T(reg) from 'day 35' autoimmune and control chimeras showed equal suppressive effects towards self-antigen-specific autoimmune T cells. Purified CD4(+)CD25(high)FR4(high) T(reg) from 'day 35' treated chimeras showed increased IL-10 and IFN-gamma mRNA-expression (RT-PCR) relative to control chimeras. In this model of CTLA-4-blockade-induced autoimmunity after allogeneic bone marrow transplantation, anti-CTLA-4-mAb gives rise to a progressive expansion - without a prior transient reduction - of T(reg) cells. T(reg) of autoimmune animals do not show a defect in in vitro suppressive function but show an in vivo activated cytokine profile, suggesting that the expansion occurs as a compensatory phenomenon to control autoimmunity.

Etanercept improves linear growth and bone mass acquisition in MTX-resistant polyarticular-course juvenile idiopathic arthritis.

OBJECTIVES: Chronic inflammation in juvenile idiopathic arthritis interferes with linear growth and bone mass acquisition. We prospectively evaluated and compared linear growth and evolution of bone mass acquisition and body composition in MTX-resistant polyarticular-course JIA (polyJIA) patients started on etanercept and in recently diagnosed polyJIA patients started on MTX monotherapy. METHODS: Sixteen MTX-resistant polyJIA patients were given add-on etanercept, eight recently diagnosed polyJIA patients were started on MTX. Patients were evaluated at baseline and at 1, 6, 12 and 18 months with respect to disease activity, linear growth, BMD and body composition. RESULTS: Baseline patient and disease characteristics were similar in both groups. Clinical disease activity (Pediatric ACR30) was equally well controlled in both groups. Growth velocity increased significantly allowing catch-up growth in the etanercept + MTX group only. BMD (lumbar spine Z-score) improved significantly in both groups. A significant increase of bone mineral content and lean:fat mass ratio was seen in the etanercept + MTX group, but not in the MTX group. CONCLUSION: Clinical control of disease activity by etanercept in MTX-refractory polyJIA is associated with rapidly instituted catch-up growth and improvement of bone mineralization and body composition. In recently diagnosed polyJIA patients treated with MTX the relation between clinical response and these parameters was less evident. Preliminary data on serum IL-6 and osteoprotegerin levels indicate that the beneficial effects seen with etanercept therapy may be related to its control of systemic IL-6 production and enhancement of osteoblast activity.

Haptoglobin deficiency facilitates the development of autoimmune inflammation.

Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL-6. HP has been demonstrated to modulate the immune response and to have anti-inflammatory activities. To analyze HP's effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp(-/-)) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). Hp(-/-)mice suffered from a more severe disease that was associated with increased expression of IL-17A, IL-6, and IFN-gamma mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL-17+ CD4+ and IFN-gamma+ CD4+ cells persisted in the CNS of Hp(-/-) mice. Absence of HP affected the priming and differentiation of T cells after MOG(35-55) immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp(-/-) T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.

ICOS deficiency results in exacerbated IL-17 mediated experimental autoimmune encephalomyelitis.

INTRODUCTION: Inducible costimulatory molecule (ICOS) is important for the effector function of T cells, especially for Th2 and T cell dependent B cell responses. However, it has been shown that ICOS is required for the differentiation of Th17 cells. Since IL-17 has been identified as a major cytokine involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the enhanced severity of EAE in ICOS-deficient mice (ICOS(-/-)) mice is unexpected. METHODS: To better understand the role of ICOS and of IL-17 in EAE, we induced EAE in ICOS(-/-) by immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) in complete Freund's adjuvant. RESULTS: As previously reported, we found that ICOS(-/-) mice developed more severe EAE. Upon restimulation with MOG(35-55,) splenocytes from ICOS(-/-) mice with EAE produced higher amounts of IL-17 and ICOS(-/-) mice had a higher expression of IL-17, IL-6, and TGF-beta mRNA in the spinal cords at the onset of the disease. Finally, the blockade of IL-17 strongly inhibited disease even in ICOS(-/-) mice, showing that IL-17 is playing a major role in the pathogenesis of EAE both in WT and ICOS(-/-) mice. CONCLUSION: In conclusion, MOG immunization induces MOG-specific Th17 cells also in ICOS(-/-) mice, and a higher expression of IL-17 and of Th17-driving cytokines IL-6 and TGF-beta in the central nervous system at the onset of EAE that correlates with their more severe disease.

Generation of antibody responses to pneumococcal capsular polysaccharides is independent of CD1 expression in mice.

Streptococcus pneumoniae is a bacterial microorganism that frequently causes serious infection, particularly in children and the elderly. Protection against infection with S. pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS), but the mechanisms responsible for the generation of anticapsular antibodies remain incompletely understood. The aim of the present study was to evaluate the role of CD1-restricted T cells in the antibody response to caps-PS. When immunized with Pneumo23, wild-type mice and CD1 knockout mice on BALB/c and C57BL/6 backgrounds generated immunoglobulin M (IgM) and IgG antibody responses to soluble caps-PS that were comparable. Similar results were obtained after immunization with heat-inactivated S. pneumoniae. The IgM and IgG antibody response of wild-type mice to Pneumo23 was not affected by an antagonizing monoclonal anti-CD1 antibody treatment. In summary, our data provide evidence that the antibody response to caps-PS is generated independently of CD1 expression.

Aprotinin reduces cardiac troponin I release and inhibits apoptosis of polymorphonuclear cells during off-pump coronary artery bypass surgery.

OBJECTIVES: In addition to blood-sparing effects, aprotinin may have cardioprotective and anti-inflammatory effects during cardiopulmonary bypass-assisted cardiac surgery. In this study, the authors examined whether aprotinin had cardioprotective and/or anti-inflammatory effects in patients undergoing off-pump coronary artery bypass grafting. DESIGN: A prospective randomized clinical trial. SETTING: University hospital. PARTICIPANTS: Fifty patients were randomized to control (n = 25) or aprotinin treatment (n = 25) groups. INTERVENTIONS: Aprotinin was given as a loading dose (2 x 10(6) KIU) followed by a continuous infusion at 5 x 10(5) KIU/h until skin closure. MEASUREMENTS AND MAIN RESULTS: Blood samples for cardiac troponin I; interleukin-6, interleukin-8, and interleukin-10; tumor necrosis factor alpha; and elastase were taken after anesthesia induction, completion of revascularization, and 6 hours, 12 hours, and 24 hours after revascularization. Blood samples were taken to assess for apoptosis in polymorphonuclear cells. Baseline plasma levels for cardiac troponin I did not differ between groups but were significantly lower in aprotinin-treated patients at the time of revascularization (p = 0.03) and 6 hours (p = 0.004) and 24 hours (p = 0.03) later. Aprotinin significantly reduced apoptosis in polymorphonuclear cells compared with control-treated patients (p = 0.04). There were no differences in plasma cytokine or elastase levels between groups. CONCLUSIONS: The authors conclude that aprotinin reduces perioperative cardiac troponin I release and attenuates apoptosis in polymorphonuclear cells but has no significant effects on plasma cytokine levels in patients undergoing off-pump coronary artery bypass graft surgery.

Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells.

Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.

Intravenous immunoglobulins in refractory childhood-onset epilepsy: effects on seizure frequency, EEG activity, and cerebrospinal fluid cytokine profile.

PURPOSE: Several studies have reported favorable effects of intravenous immunoglobulins (IVIG) in refractory epilepsy. Evidence substantiating an immunomodulatory action is scarce. In an open-label study, we prospectively investigated the effect of IVIG on clinical, EEG and serum/CSF immunological parameters in patients with refractory childhood-onset epilepsy. METHODS: Thirteen patients (median age 6.9 years; range 1.6-25.8) with refractory seizures despite 3-4 antiepileptic drug regimens were given IVIG (Sandoglobulin, ZLB-Behring, add-on, 4 x 400 mg/kg/3 weeks). Seizure frequency, 24-h video-EEG, and CSF/serum immunological parameters and cytokine profiles (IL-6/IL-8/IL-12/IL-10) were documented before and after completion of the course. RESULTS: Seizure frequency was reduced by > or = 50% in four, and by 25%-50% in three patients. In contrast, variation in automatically recorded spike counts (1-h-wake and -sleep) did not correlate with clinical improvement. Serum immunological parameters showed variable deviations in eight patients (e.g., IgG(2) deficiency) and CSF immunoblotting showed oligoclonal bands in two patients. Blood-brain barrier permeability was normal in 12 patients. IL-6 and IL-8 were clearly detectable in CSF of all patients; the levels were significantly higher than those in plasma but remained unaffected by IVIG treatment. CONCLUSIONS: Despite unchanged EEG spike counts, substantial reductions in seizure frequency occurred in 7 of 13 patients, suggesting that IVIG hinder progression of central epileptic activity into clinical seizures. Intrathecal presence of IL-8 and IL-6 was documented in all patients, but was unaffected by IVIG, suggesting that their production is directly related to electrical seizure activity and that IVIG may act through interference with immune pathways downstream to IL-6 and IL-8.

Acute inflammatory and anabolic systemic responses to peak and constant-work-rate exercise bout in hospitalized patients with COPD.

STUDY OBJECTIVES: To explore the acute systemic inflammatory and anabolic effects of cycling in hospital admitted patients with chronic obstructive pulmonary disease (COPD) and in patients with clinically stable disease. DESIGN: Cross-sectional comparative study. SETTING: University Hospital Gasthuisberg, a tertiary care setting. PATIENTS: 16 patients with clinically stable COPD (no acute exacerbation in the past 12 weeks; median age: 73 years (IQR: 60 to 75); median forced expiratory volume in the first second (FEV1): 45% predicted (IQR: 33 to 58)) and 14 patients who were admitted to a hospital due to an acute exacerbation of COPD (median age: 65 years (IQR: 59 to 74); median FEV1 on day 8 of hospital stay: 41% predicted (IQR: 33 to 54)). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Circulating levels of C reactive protein, interleukin 6, interleukin 8 and insulin-like growth factor I were determined before, at the end and 2 and 30 minutes after a symptom-limited peak cycling test and before, at the end and 2 and 30 minutes after a symptom-limited constant-work-rate cycling test at 70% of the peak load. Non-significant changes in the circulating markers of inflammation and anabolism were found during or up to 30 minutes after ceasing the peak or constant-work-rate cycling exercise tests. The systemic responses of the hospitalized patients with COPD did not differ from those with clinically stable disease. CONCLUSIONS: High-intensity cycling exercises did not increase the circulating levels of inflammatory markers in patients with chronic obstructive pulmonary disease, irrespective of their clinical stability.

Immature and mature monocyte-derived dendritic cells in myelodysplastic syndromes of subtypes refractory anemia or refractory anemia with ringed sideroblasts display an altered cytokine profile.

Dendritic cells (DC) are pivotal for T cell-mediated immunity. We investigated the early and terminal maturation of monocyte-derived DC (MoDC) in myelodysplastic syndromes (FAB subtypes refractory anemia (MDS-RA) or refractory anemia with ringed sideroblasts (MDS-RARS)). Immature MoDC were obtained by culture of monocytes with GM-CSF and IL-4 for 8 days. To obtain mature MoDC, TNF-alpha was added during the final three culture days. T cell proliferation and T cell cytokine secretion in mixed lymphocyte reactions (MLR) unveiled a strong reduction of allostimulatory capacity of mature but also of immature MoDC from MDS patients. Immature MoDC from MDS patients exhibited an almost normal immunophenotype, but secreted substantially less IL-12 and more IL-10 in response to LPS/IFN-gamma than normal controls. Terminal addition of TNF-alpha to GM-CSF/IL-4 treated monocytes failed to extinguish cytokine production by MDS MoDC and failed to induce the expected membrane upregulation of costimulatory and other ligands as in normal controls. While our data provide further support for previous studies that have indicated an impaired differentiation of immature towards mature MoDC, they also clearly demonstrate a qualitatively and quantitatively altered cytokine secretion at the level of immature MoDC, which may in part explain the reduced allostimulatory capacity of these cells. These alterations may contribute to immune modulation of the clinical phenotype of marrow failure in MDS, and may have to be considered when designing DC-based immunotherapeutic strategies for MDS.

Immunosuppressive activity of a new pteridine derivative (4AZA1378) alleviates severity of TNBS-induced colitis in mice.

Besides TNF, activated T cells play a central role in the pathogenesis of inflammatory bowel diseases such as Crohn's disease. New therapies are still awaited to cure these often debilitating diseases. Natural occurring pteridines such as tetrahydrobiopterin (BH4) and neopterin have been reported to have immune modulating activities. Starting from a pteridine scaffold library, we intended to select compounds with potent in vitro inhibitory effects on T cells and to evaluate in vivo efficacy of selected compounds on trinitrobenzenesulphonate (TNBS) colitis in mice. Compound 4AZA1378 was selected because it potently inhibits human T cell proliferation at low nM concentrations (IC50 4 nM) while an almost 50-fold higher concentration was needed to inhibit LPS-induced TNF production. Mice treated with 4AZA1378 had less severe signs of colitis after TNBS rectal administration, with a more rapid weight recovery. Myeloperoxidase (MPO) activity and intralesional cytokine production were lower in mice of the treated groups. Furthermore anti-TNBS antibody responses were completely inhibited by treatment with 4AZA1378. In conclusion, we identified a pteridine analogue 4AZA1378 with immunosuppressive activity and a strong remission-inducing effect in TNBS colitis, supporting further pre-clinical and clinical development of this novel molecule for treatment of inflammatory diseases.

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling.

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.

Anti-inflammatory activity of a pteridine derivative (4AZA2096) alleviates TNBS-induced colitis in mice.

Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis and Crohn's disease. Naturally occurring pteridine analogs have been reported to have potent immunomodulatory activity, especially on TNF production. The aim of this study is to identify small molecule TNF inhibitiors derived from pteridine and to prove their in vivo efficacy in an inflammatory model. A focused chemical library based on the pteridine scaffold was screened in vitro on lipopolysaccharide (LPS)-induced TNF production in peripheral blood mononuclear cells (PBMC). One synthetic pteridine analog (4AZA2096), shown to have strong inhibitory activity, was selected and tested for its efficacy to treat trinitrobenzenesulfonate (TNBS)-induced colitis in mice, a model of Crohn's disease. Colitis was induced by rectal administration of 1 mg TNBS in 50% ethanol after presensitization via the skin. The synthetic pteridine analog 4AZA2096 was shown to potently inhibit LPS-induced TNF production in vitro. Colitic mice treated with 4AZA2096 orally (20 mg/kg/day) recovered more rapidly and, histologically, had a reduction of inflammatory lesions, less edema, a reduction of goblet cell loss, and reduced wall thickness. Cell infiltration in the colon, especially infiltration of neutrophils, as shown by myeloperoxidase (MPO) activity, was reduced in 4AZA2096-treated animals. Intralesional TNF production was lower in mice of the treated groups, whereas interleukin-18 (IL-18) and interferon-gamma (IFN-gamma) mRNA were not affected. Treatment had no effect on anti-TNBS antibody production, arguing against generalized immunosuppression. In conclusion, we identified a pteridine derivative, 4AZA2096, with strong inhibitory activity on TNF production and a remission- inducing effect in TNBS colitis, supporting further preclinical and clinical development of this novel TNF inhibitor for treatment of inflammatory diseases.

Noggin haploinsufficiency differentially affects tissue responses in destructive and remodeling arthritis.

OBJECTIVE: The balance between destruction and homeostatic or reparative responses determines the outcome of arthritis. Increasing evidence suggests a role for signaling pathways, essential for development and growth, in the maintenance of tissue homeostasis and attempts at repair. Inappropriate activation of such pathways may also have a role in disease progression. We undertook this study to determine the effect of shifting the balance in bone morphogenetic protein (BMP) signaling in different mouse models of arthritis. METHODS: Endogenous levels of noggin, a BMP antagonist, were reduced using heterozygous noggin(+/LacZ) mice in a model of inflammation-driven destruction (methylated bovine serum albumin [mBSA]-induced monarthritis), a model of systemic autoimmune arthritis (collagen-induced arthritis [CIA]), and a model of joint ankylosis (spontaneous arthritis in DBA/1 mice). In addition, we studied BMP inactivation by adenoviral noggin overexpression in destructive arthritis. Cartilage damage and activation of BMP signaling were studied by digital image analysis using Safranin O sulfated glycosaminoglycan staining and immunohistochemistry for phosphorylated Smads (Smads 1, 5, and 8), respectively. RESULTS: Noggin haploinsufficiency provided protection for articular cartilage against destruction in mBSA-induced arthritis. Antagonist overexpression rendered cartilage more vulnerable in this model. Noggin gene transfer in knees affected by CIA also enhanced cartilage damage. Haploinsufficiency did not affect CIA, but noggin(+/LacZ) mice had an increased number of CD4-positive cells with normal immune responses. In noggin(+/LacZ) DBA/1 mice with spontaneous arthritis, we observed delayed progression from cartilage to bone formation. CONCLUSION: Tight spatiotemporal control of BMP signaling appears to be critical in the response of joint tissues in models of arthritis.