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.Cytokines are soluble proteins that are secreted by cells of the immune system. These proteins can alter the behavior and properties of different cell types. Although cytokine functions are complex, cytokine profiles are highly relevant parameters of an immune response. Different cytokines possess biological overlapping functions, and they have the ability to regulate production of other cytokines. Therefore, analysis of the function of the complete set of cytokines expressed within microenvironments (e.g., a site of inflammation) is often of more value than analysis of a single isolated cytokine (13).Cytokines can be quantitated at various levels. mRNA can be detected by real-time PCR; intracellular proteins can be measured by fluorescence-activated cell sorter staining of permeabilized cells, and secreted cytokines can be quantified with bioassays, enzyme-linked immunosorbent assays (ELISAs), radioactive immunosorbent assays, and microarrays. Multiplex assays for detection of cytokines at the mRNA (6) and cellular levels (16, 18) are commonly used. However, these assays have one or more limitations, like the need for a large sample volume or detection of precursor proteins rather than native secreted proteins. In addition, these techniques are time-consuming and laborious.Recent advances concerning applications for the simultaneous detection of proteins have resulted in different particlebased flow cytometric assays. These assays have proven to be very useful in the simultaneous detection of cytokines in body fluids. Unfortunately, at present, either the number of different microspheres or the availability of predefined kits limits these assays (1, 3). The Bio-Plex system employing the Luminex multianalyte profiling technology (Lab-MAP) allows individual and multiplex analysis of up to 100 different analytes in a single microtiter well (20).Our laboratory focuses on immunoregulation and immunotherapy of children with autoimmune diseases-in particular, juvenile idiopathic arthritis (JIA). Sample volumes are relatively small due to our patient population. For a number of cytokines, ready-to-use beads are available, but not for the full spectrum. To overcome these limitations, we chose to develop and validate our own multiplex assays with the Bio-Plex system. With this assay, we were able to detect human cytokines in antigen-stimulated peripheral blood mononuclear cell (PBMC) culture supernatants from both autoimmune and healthy individuals. We showed that it is a reliable, fast, and reproducible technique with a sensitivity that is comparable to that of conventional ELISAs.
MATERIALS AND METHODSCell isolation and cultures. Heparinized blood ...
Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal dismay and skin rash. Diagnostic hallmark of HIDS is a constitutively elevated level of serum immunoglobulin D (IgD), although patients have been reported with normal IgD levels. To determine the underlying defect in HIDS, we analysed urine of several patients and discovered increased concentrations of mevalonic acid during severe episodes of fever, but not between crises. Subsequent analysis of cells from four unrelated HIDS patients revealed reduced activities of mevalonate kinase (MK; encoded by the gene MVK), a key enzyme of isoprenoid biosynthesis. Sequence analysis of MVK cDNA from the patients identified three different mutations, one of which was common to all patients. Expression of the mutant cDNAs in Escherichia coli showed that all three mutations affect the activity of the encoded proteins. Moreover, immunoblot analysis demonstrated a deficiency of MK protein in patient fibroblasts, indicating a protein-destabilizing effect of the mutations.
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.
MRP8 and MRP14 are specifically released during the interaction of monocytes with inflammatory activated endothelium, probably at sites of local inflammation. Their serum concentrations represent a useful marker for monitoring local inflammation in JRA.
Objective. To conduct a prospective cohort study using anakinra, a recombinant IL-1 receptor antagonist (IL-1Ra), as first-line therapy in patients with newonset systemic juvenile idiopathic arthritis (JIA). Results. We included consecutive patients with new-onset systemic JIA. The mean followup period was 32 months (range 12-54 months). At the 3-month time point, 85% of the patients showed an adapted ACR Pedi 90 response or had inactive disease; 75% of the patients achieved this response while receiving recombinant IL-1Ra alone. After 1 year, 17 of the 20 patients met the criteria for clinically inactive disease, and 13 of these patients met these criteria while receiving monotherapy with recombinant IL-1Ra. However, because of persistent disease activity, 7 of the 20 patients required additional therapy besides recombinant IL-1Ra. According to our stop strategy, 73% of patients with at least an adapted ACR Pedi 90 response at 3 months could stop recombinant IL-1Ra treatment within 1 year. After 2 years, 12 (86%) of 14 patients met the criteria for disease remission, either while receiving (n ؍ 4) or not receiving (n ؍ 8) medication. After 3 years, 10 (91%) of 11 patients met the criteria for disease remission, either while receiving (n ؍ 2) or not receiving (n ؍ 8) medication.Conclusion. This is the first prospective study in which recombinant IL-1Ra was used as first-line therapy in patients with systemic JIA. We observed excellent responses in nearly all patients within 3 months. In the majority of responding patients, treatment with recombinant IL-1Ra could be stopped within 1 year, with remission being preserved during followup. In approximately one-third of patients, concomitant therapy was required for maintenance of clinical response.Systemic juvenile idiopathic arthritis (JIA) is a severe subtype of JIA that accounts for ϳ10% of JIA cases (1).
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