J. van der Ven-Jongekrijg

Cytokine patterns in patients after major vascular surgery, hemorrhagic shock, and severe blunt trauma. Relation with subsequent adult respiratory distress syndrome and multiple organ failure.

ObjectiveThis study investigates the course of serum cytokine levels in patients with multiple trauma, patients with a ruptured abdominal aortic aneurysm (AAA), and patients undergoing elective AAA repair and the relationship of these cytokines to the development of adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Summary Background DataSevere tissue trauma, hemorrhagic shock, and ischemia-reperfusion injury are pathophysiologic mechanisms that may result in an excessive uncontrolled activation of inflammatory cells and mediators. This inflammatory response is thought to play a key role in the development of (remote) cell and organ dysfunction, which is the basis of ARDS and MOF. MethodsThe study concerns 28 patients with multiple trauma, 20 patients admitted in shock because of a ruptured AAA, and 18 patients undergoing elective AAA repair. Arterial blood was serially sampled from admission (or at the start of elective operation) to day 13 in the intensive care unit, and the serum concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were determined. ResultsTwenty-two patients died, 15 within 48 hours and 7 after several weeks, as a result of ARDS/MOF. At hospital admission and after 6 hours, these nonsurvivors had significantly higher plasma TNF-α and IL-1β levels than did the survivors. At the same measuring points, TNF-α and IL-1β were significantly more elevated in patients with ruptured AAA than in traumatized patients. However, IL-6 was significantly higher in the traumatized patients. In 10 patients, ARDS/MOF developed, and 41 had an uncomplicated course in this respect. Those with ARDS/MOF exhibited significantly different cytokine patterns in the early postinjury phase. TNF-α and IL-1β levels were higher mainly on the first day of admission; IL-6 concentrations were significantly elevated in patients with ARDS/MOF from the second day onward. The latter cytokine showed a good correlation with the daily MOF score during the whole 2-week observation paeriod. ConclusionsIn the early postinjury phase, higher concentrations of these cytokines are associated; not only with an increased mortality rate, but also with an increased risk for subsequent ARDS and MOF. These data therefore support the concept that these syndromes are caused by an overwhelming autodestructive inflammatory response.

Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year

Dietary supplementation with n‐3 fatty acids from fish oil alleviates inflammation in various chronic inflammatory disease states. Reductions in the production of pro‐inflammatory cytokines interleukin 1β (IL‐1β), tumour necrosis factor alpha (TNF‐α), and interleukin 6 (IL‐6) have been seen in humans after short‐term n‐3 fatty acid supplementation. We investigated long‐term effects of dietary n‐3 fatty acids on circulating cytokine concentrations and on ex vivo stimulated whole‐blood production of IL‐1β, TNF‐α and interleukin 1 receptor antagonist (IL‐1Ra), the naturally occurring antagonist of IL‐1. A total of 58 monks with a mean age of 56 years were randomized into four groups and their diets were supplemented with 0, 3, 6, or 9 g of fish oil, providing 0, 1.06, 2.13 or 3.19 g of n‐3 fatty acids per day. Subjects received equal amounts of saturated fatty acids, vitamin E and cholesterol. Compliance was excellent and erythrocyte fatty acid profiles closely reflected the amounts of n‐3 fatty acids ingested. In the group receiving 9 g of fish oil per day, no influence of n‐3 fatty acids on circulating cytokine concentrations was observed relative to placebo. Endotoxin‐stimulated whole‐blood cytokine production was measured at 26 and 52 weeks after the start and at 4, 8 and 26 weeks after cessation of supplementation. In all groups, the production of IL‐1β and IL‐1Ra was higher during supplementation than afterwards. However, no differences in cytokine production were noted between the placebo group and the various treatment groups at any point in time. Our results suggest that long‐term supplementation of fish oil does not affect ex vivo cytokine production in man.

Sustained efficacy of the monoclonal anti-interleukin-1 beta antibody canakinumab in a 9-month trial in Schnitzler's syndrome

Objectives Schnitzlers syndrome is a chronic disabling autoinflammatory disorder, characterised by chronic urticaria, paraproteinemia and systemic inflammation. The interleukin (IL) 1 receptor antagonist anakinra is a very effective treatment, but requires daily injection and blocks both IL-1α and IL-1β. Canakinumab is a selective human monoclonal anti-IL-1β antibody with a long half-life. We investigated the long-term efficacy and safety of canakinumab in Schnitzlers syndrome. Methods In an open-label, single-treatment arm trial, eight patients with Schnitzlers syndrome received monthly injections with 150 mg canakinumab subcutaneously for 6 months, followed by a 3-month observation period. Primary outcome was complete or clinical remission at day 14. Secondary outcome measures included inflammatory markers, quality of life, time to relapse, safety and tolerability. Results After stopping anakinra, patients developed moderate to severe clinical symptoms. Canakinumab induced complete or clinical remission at day 14 in all eight patients. Median C-reactive protein concentrations decreased from 169 mg/l at baseline to less than 10 mg/l on day 14 and remained low or undetectable. One patient discontinued participation on day 39 because of return of symptoms while all others remained in complete or clinical remission during the 6-month treatment period. Relapse after last canakinumab dose occurred within 3 months in four patients. For two patients, remission continued several months post-study. Five patients reported at least one adverse event, predominantly mild upper respiratory tract infections. One patient died in a traffic accident. Conclusions In this 9-month study, monthly 150 mg canakinumab injection was an effective and well-tolerated treatment for Schnitzlers syndrome. Our data demonstrate that IL-1β plays a pivotal role in this disease. ClinicalTrials.gov NCT01276522.

Reduced production of immunoregulatory cytokines in vitamin A- and zinc-deficient Indonesian infants

Objective: To determine effects of vitamin A, zinc and iron deficiency in Indonesian infants on the ability to produce immunoregulatory cytokines.Design, setting and subjects: Immunological asssessment was done in 59 infants participating in a cross-sectional nutritional survey in rural West Java, Indonesia. Production of T-helper cell type-1 (Th1, cell-mediated) cytokines interferon-γ (IFN-γ), interleukin-12 (IL-12), interleukin-18 (IL-18) and T-helper cell type-2 (Th2, humoral) cytokine interleukin-6 (IL-6) were measured after stimulation with lipopolysaccharide and phytohemagglutinin in an ex vivo whole blood culture system. Circulating neopterin concentrations were determined as an indicator of in vivo macrophage activity.Results: Of the infants, 48% were vitamin A deficient, 44% were anemic (with 17% having iron deficiency anemia), and 17% were zinc deficient. Vitamin-A deficient infants had significantly reduced ex vivo production of IFN-γ, but also significantly higher circulating neopterin concentrations. Production of IFN-γ and IL-12 were strongly correlated, IFN-γ and IL-18 production were not. Zinc deficiency was accompanied by significantly reduced white blood cell counts and reduced ex vivo production of IL-6. Iron status was not related to cytokine production.Conclusions: This study shows that in vitamin A deficiency there is Th1 dominance in a steady state, combined however with impairment of the Th1 response after stimulation, whereas in zinc deficiency, there is a decreased Th2 response. Overall, vitamin A deficiency and zinc deficiency have marked albeit different effects on the immunocompetence of infants, affecting both cell-mediated and humoral components of the immune system.Sponsorship: Netherlands Foundation for the Advancement of Tropical Research (WOTRO), and Ter Meulen Fund (Royal Netherlands Academy of Arts and Sciences).

Disease-specific ex vivo stimulation of whole blood for cytokine production : applications in the study of tuberculosis

As a simple method for field studies to assess the cytokine-status of patients with tuberculosis (TB), the use of whole blood instead of isolated cells has advantages, especially since the risk of contamination is minimal. Therefore, cytokine production in whole blood cultures was determined using non-specific and disease-specific stimuli. Heparinized blood from healthy volunteers was either incubated in closed vacutainer tubes or in tissue culture wells after dilution in culture medium. Dose-response and kinetics were investigated for the production of TNFalpha, IL-1beta, IL-1ra, IL-10 and IFNgamma. Patients with TB and healthy individuals were examined for IFN-gamma production in whole blood. In the absence of a stimulus, the production of cytokines is negligible in whole blood cultures. LPS induces the production of TNFalpha, IL-1beta, IL-1ra and IL-10; PHA induces the production of IFNgamma and IL-10. Live BCG induces the production of proinflammatory cytokines, irrespective of tuberculin skin status. In contrast, PPD and MTB-culture filtrate induce production of IFNgamma in skin-test positive and not in skin-test negative healthy subjects. Five out of 13 patients with TB had a low antigen-specific IFNgamma production, suggestive of a minimal or absent specific T-cell response. For most purposes, cultures in closed vacutainer tubes are optimal. If one wishes to focus on T-cell cytokines or if only small volumes of blood are available, dilution of whole blood in culture medium before incubation in tissue culture wells may be preferable.

Macrophage migration inhibitory factor (MIF) in meningococcal septic shock and experimental human endotoxemia.

Macrophage migration inhibitory factor (MIF) is a mediator of innate immunity and important in the pathogenesis of septic shock. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF) &agr; are reported to be inducers of MIF. We studied MIF and cytokines in vivo in patients with meningococcal disease, in human experimental endotoxemia, and in whole blood cultures using a newly developed sensitive and specific enzyme-linked immunosorbent assay. Twenty patients with meningococcal disease were investigated. For the human endotoxemia model, 8 healthy volunteers were intravenously injected with 2 ng/kg Escherichia coli LPS. Whole blood from healthy volunteers was incubated with LPS or heat-killed meningococci. Macrophage migration inhibitory factor concentration in blood was increased during meningococcal disease and highest in the patients presenting with shock compared with patients without shock. Plasma concentration of MIF correlated with disease severity, the presence of shock and with the cytokines interleukin (IL) 1&bgr;, IL-10, IL-12, and vascular endothelial growth factor, but not with TNF-&agr;. MIF was not detected in blood in experimental endotoxemia, nor after stimulation of whole blood with LPS or meningococci, although high levels of TNF-&agr; were seen in both models. In conclusion, MIF is increased in patients with meningococcal disease and highest in the presence of shock. Macrophage migration inhibitory factor cannot be detected in a human endotoxemia model and is not produced by whole blood cells incubated with LPS or meningococci.

The effects of dexamethasone and chlorpromazine on tumour necrosis factor‐α, interleukin‐1β, interleukin‐1 receptor antagonist and interleukin‐10 in human volunteers

Tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) are pro‐inflammatory cytokines that play an important role in severe infections, whereas IL‐1 receptor antagonist (IL‐1ra) and IL‐10 are anti‐inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF‐α and IL‐1β. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)‐stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time‐points after medication. Circulating cytokine concentrations were low in all samples. LPS‐induced TNF‐α and IL‐1β production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1–100 ng/ml) had no modulatory action on TNF‐α, IL‐1β, IL‐1ra or IL‐10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF‐α and IL‐1β production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti‐cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro‐inflammatory cytokine production.

THU0377 Efficacy, safety and pharmacokinetics of the anti-interleukin-1 beta antibody canakinumab in patients with schnitzler syndrome

Background Schnitzler syndrome is a chronic disabling autoinflammatory disorder, characterized by chronic urticaria, paraproteinemia and systemic inflammation. The interleukin-1 receptor antagonist (IL-1Ra) anakinra has been reported as very effective, but requires daily injection because of its short half-life, and is associated with injection site reactions. Canakinumab is a fully human monoclonal selective anti-IL-1β antibody with a half-life of ∼28-30 days in adults with CAPS.1 Objectives To investigate the efficacy, safety and drug dynamics of canakinumab in Schnitzler syndrome patients. Methods Eight adults with active classical or variant type Schnitzler syndrome who previously responded to anakinra, entered a 9-month trial comprising 6 months of open-label 150mg canakinumab sc injections every 4 weeks and 3 months follow-up. Patients were evaluated clinically using 5-point Physician and Patient Global Assessment of disease activity scales and serologically by C-reactive protein (CRP) levels. The primary endpoint was the percentage of patients with complete or clinical remission at Day 14. Complete remission was defined as clinical remission with normal CRP and clinical remission was defined as having absent or minimal disease activity and >70% improved, but still elevated, CRP. Canakinumab disposition was characterized using pharmacokinetic modeling methodologies from blood samples collected throughout the trial. Results Upon discontinuation of anakinra, all patients developed active Schnitzler syndrome characterized by general malaise, non-pruritic to burning urticaria, fever and arthralgia or arthritis. In all patients, complete or clinical remission was achieved at Day 14. Mean CRP concentrations decreased from 162 at baseline to <10 mg/L on Day 14 and remained low or undetectable afterwards. One patient discontinued on Day 39 because of return of symptoms while all other patients remained in complete or clinical remission for the remainder of the 6-month treatment period. Relapse in the follow-up period occurred in 4 patients and ranged between 1.5 and 3.5 months after the last canakinumab dose. For 2 patients, remission is continuing >6 months post-study to date. Five patients reported at least 1 adverse event with only rhinitis, pharyngitis and vertigo reported in >1 patient (N=2). One mild local site reaction was reported and 1 patient died in a traffic accident (Day 185). Preliminary analysis of pharmacokinetics results are comparable to the results obtained in previous trials in CAPS patients. Conclusions In this 9-month study, monthly 150mg canakinumab injection was an effective and well-tolerated treatment in Schnitzler syndrome. Adverse events were manageable. Based on preliminary modeling, PK of canakinumab in patients with Schnitzler syndrome was similar to that in CAPS patients. Data suggest that IL-1β plays a pivotal role in this disease and further study is needed to better define canakinumab treatment. References Lachmann HJ, Kone-Paut I, Kuemmerle-Deschner JB et al. Use of canakinumab in the cryopyrin-associated periodic syndrome. N Engl J Med 2009; 360(23):2416-2425. Disclosure of Interest H. De Koning Grant/Research support from: Novartis, J. Schalkwijk: None Declared, J. van der Ven-Jongekrijg: None Declared, M. Stoffels: None Declared, J. van der Meer Consultant for: Novartis, A. Simon Grant/Research support from: Novartis, Consultant for: Novartis