Wietse Kuis

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

ABSTRACT Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.

CD4+CD25bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis.

This study investigates the role of CD4+CD25+ regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4+CD25bright T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4+CD25bright T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4+CD25int T cells and therefore of CD4+CD25total in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4+CD25bright T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4+CD25int T cell population. The CD4+CD25bright cells of both patient groups and the CD4+CD25int cells of pers-OA JIA patients were able to suppress responses of CD25neg cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4+CD25bright T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4+CD25+ Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4+CD25bright T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4+CD25bright T cells. These data suggest that CD4+CD25bright Treg cells play a role in determining the patient’s fate toward either a favorable or unfavorable clinical course of disease.

Effect of conjugate pneumococcal vaccine followed by polysaccharide pneumococcal vaccine on recurrent acute otitis media: a randomised study.

BACKGROUND Pneumococcal conjugate vaccine prevents recurrent acute otitis media (AOM) in infants immunised at 2, 4, 6, and 12-15 months of age. We aimed to find out whether this vaccine also prevents AOM in older children who have had previous episodes of AOM. METHODS In this double-blind, randomised study, we enrolled 383 patients aged 1-7 years who had had two or more episodes of AOM in the year before entry. Randomisation was stratified in four groups according to age (12-24 months vs 25-84 months) and the number of previous AOM episodes (two or three episodes vs four or more episodes). Children received either 7-valent pneumococcal conjugate vaccine followed by 23-valent pneumococcal polysaccharide vaccine, or hepatitis A or B vaccines. They were followed up for 18 months for recurrence of AOM. We also cultured samples of middle-ear fluid and nasopharyngeal swabs to assess association of pneumococcal serotypes with AOM after vaccination. FINDINGS We noted no reduction of AOM episodes in the pneumococcal vaccine group compared with controls (intention-to-treat analysis: rate ratio 1.25, 95% CI 0.99-1.57). Although nasopharyngeal carriage of pneumococci of serotypes included in the conjugate-vaccine was greatly reduced after pneumococcal vaccinations, immediate and complete replacement by non-vaccine pneumococcal serotypes took place. INTERPRETATION These data do not lend support to the use of pneumococcal conjugate vaccine to prevent otitis media in previously unvaccinated toddlers and children with a history of recurrent AOM.

Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study

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 20 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 α, 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. Conclusion: 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.

Autologous haemopoietic stem-cell transplantation in four patients with refractory juvenile chronic arthritis

BACKGROUND Autologous haemopoietic stem-cell transplantation (AHSCT) had been described as a possible treatment for severe autoimmune disease refractory to conventional treatment. We report the first four children with severe forms of juvenile chronic arthritis (JCA) treated with AHSCT. METHODS We studied three children with systemic JCA and one child with polyarticular JCA. Unprimed bone marrow was taken 1 month before AHSCT. T-cell depletion of the graft was done with CD2 and CD3 antibodies. We used a preparative regimen of antithymocyte globulin (20 mg/kg), cyclophosphamide (200 mg/kg) and low-dose total body irradiation (4 Gy). Methotrexate and cyclosporin were stopped before AHCST, prednisone was tapered after 2 months. FINDINGS Our patients showed a drug-free follow-up of 6-18 months with a marked decrease in joint swelling, pain, and morning stiffness. Erythrocyte sedimentation rate, C-reactive protein, and haemoglobin returned to almost normal values within 6 weeks. Despite T-cell depletion there was a rapid immune reconstitution in three out of four children. Two patients developed a limited varicella zoster virus eruption, which was treated by aciclovir. INTERPRETATION AHSCT for severe JCA was well tolerated and induced a remission of disease in four children with JCA that was resistant to conventional treatment. Prolonged prednisone-free growth catch-up and general well-being is a major therapeutic gain in such children. The actual follow-up is too short, however, for us to conclude that these children are completely cured of their disease.

Tolerogenic immune responses to novel T-cell epitopes from heat-shock protein 60 in juvenile idiopathic arthritis

BACKGROUND Juvenile idiopathic arthritis is a heterogeneous autoimmune disease characterised by chronic inflammation of one or more joints. In patients with this disease, T-cell reactivity to autologous heat-shock protein 60 (HSP60) is associated with a favourable prognosis. We sought to identify HSP60 T-cell epitopes to find potential targets for HSP60 immunotherapy and to assess whether immune responses to these epitopes contribute to the distinct clinical outcome of this disease. METHODS We identified eight potential epitopes using a computer algorithm from both self and microbial HSP60 binding to many HLA-DR molecules. We analysed the pattern of T-cell responses induced by these HSP60 peptides in peripheral-blood mononuclear cells (PBMC) of 57 patients with juvenile idiopathic arthritis, 27 healthy controls, and 20 disease controls. We undertook in-vitro MHC binding studies with the identified peptides, and HLA class II typing of a subset of patients with juvenile idiopathic arthritis. FINDINGS Five of the eight peptides identified yielded proliferative T-cell responses in 50-70% of PBMC from patients with juvenile idiopathic arthritis irrespective of MHC genotype, but not in PBMC from healthy or disease controls. Although PBMC from both patients with juvenile idiopathic arthritis and healthy controls produced interferon gamma in response to these peptides, only PBMC from patients with the disease produced interleukin 10. INTERPRETATION The recorded T-cell-induction in juvenile idiopathic arthritis is tolerogenic. In patients with oligoarticular disease, the immune responses to the HSP60 epitopes identified could contribute to disease remission. RELEVANCE TO PRACTICE The broad recognition of these HSP60 epitopes in a population of patients with polymorphic MHC genotypes opens the way for HSP60-peptide immunotherapy, representing a novel treatment option to specifically modulate the immune system in patients with juvenile idiopathic arthritis.

Swaddling: A Systematic Review

Swaddling was an almost universal child-care practice before the 18th century. It is still tradition in certain parts of the Middle East and is gaining popularity in the United Kingdom, the United States, and the Netherlands to curb excessive crying. We have systematically reviewed all articles on swaddling to evaluate its possible benefits and disadvantages. In general, swaddled infants arouse less and sleep longer. Preterm infants have shown improved neuromuscular development, less physiologic distress, better motor organization, and more self-regulatory ability when they are swaddled. When compared with massage, excessively crying infants cried less when swaddled, and swaddling can soothe pain in infants. It is supportive in cases of neonatal abstinence syndrome and infants with neonatal cerebral lesions. It can be helpful in regulating temperature but can also cause hyperthermia when misapplied. Another possible adverse effect is an increased risk of the development of hip dysplasia, which is related to swaddling with the legs in extension and adduction. Although swaddling promotes the favorable supine position, the combination of swaddling with prone position increases the risk of sudden infant death syndrome, which makes it necessary to warn parents to stop swaddling if infants attempt to turn. There is some evidence that there is a higher risk of respiratory infections related to the tightness of swaddling. Furthermore, swaddling does not influence rickets onset or bone properties. Swaddling immediately after birth can cause delayed postnatal weight gain under certain conditions, but does not seem to influence breastfeeding parameters.

Functional α1-adrenergic receptors on leukocytes of patients with polyarticular juvenile rheumatoid arthritis

During the last decade it has been shown that the central nervous system can influence the immune system. In healthy individuals, catecholamines can inhibit the production of pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) via interaction with beta 2-adrenergic receptors. In contrast, we show here that catecholamines can stimulate the production of the interleukin-6 (IL-6) in children with the chronic inflammatory disease polyarticular juvenile rheumatoid arthritis (JRA). The induction of IL-6 is mediated by triggering of alpha 1-adrenergic receptors on peripheral blood leucocytes of the patients with polyarticular JRA. Functional alpha 1-adrenergic receptors are absent on leukocytes of normal donors and on leukocytes of patients with the oligoarticular form of the disease.

Disturbed Neuroendocrine-Immune Interactions in Chronic Fatigue Syndrome

The present study was designed to investigate the interaction between neuroendocrine mediators and the immune system in chronic fatigue syndrome (CFS). We examined the sensitivity of the immune system to the glucocorticoid agonist dexamethasone and the beta2-adrenergic agonist terbutaline in 15 adolescent girls with CFS and 14 age- and sex-matched controls. Dexamethasone inhibits T-cell proliferation in healthy controls and in CFS patients. However, the maximal effect of dexamethasone on T-cell proliferation is significantly reduced in CFS patients as compared with controls. The beta2-adrenergic receptor agonist terbutaline inhibits tumor necrosis factor-alpha production and enhances interleukin-10 production by monocytes. Our data demonstrate that the capacity of a beta2-adrenergic agonist to regulate the production of these two cytokines is also reduced in CFS patients. We did not observe differences in baseline or CRH-induced cortisol and ACTH between CFS patients and controls. Baseline noradrenaline was similar in CFS and controls, whereas baseline adrenaline levels were significantly higher in CFS patients. We conclude that CFS is accompanied by a relative resistance of the immune system to regulation by the neuroendocrine system. Based on these data, we suggest CFS should be viewed as a disease of deficient neuroendocrine-immune communication.

Incidence and hemodynamic characteristics of near-fainting in healthy 6- to 16-year old subjects

OBJECTIVES We studied the incidence and hemodynamic characteristics of near-fainting under orthostatic stress in healthy children and teenagers. BACKGROUND Orthostatic stress testing is increasingly used to identify young subjects with unexplained syncope. However, the associated incidence of syncope and hemodynamic responses in normal young subjects are not well known. METHODS Eighty-four healthy subjects 6 to 16 years old performed forced breathing, stand-up and 70 degrees tilt-up tests. An intravenous line to sample blood for biochemical assessment of sympathetic function was introduced between the stand-up and tilt-up tests. Finger arterial pressure was measured continuously. Left ventricular stroke volume was computed from the pressure pulsations. RESULTS Sixteen of the 84 subjects were excluded because of technical problems. The incidence of a near-fainting response in the remaining 68 subjects was 10% (7 of 68) for the stand-up test and 40% (29 of 68) for the tilt-up test. Baseline parasympathetic and sympathetic activity of nonfainting and near-fainting subjects was not different. Near-fainting was characterized by attenuated systemic vasoconstriction and exaggerated tachycardia that occurred as early as 1 min after return to the upright position. On tilt-up, plasma adrenaline levels increased by a factor of 2, with slightly higher increments in the near-fainting subjects. CONCLUSIONS Inadequate vasoconstriction is the common underlying mechanism of near-fainting in young subjects. The remarkably high incidence of near-fainting during the tilt-up test after intravascular instrumentation raises serious doubts about the utility of this procedure in evaluating syncope of unknown origin in young subjects.