Selection of perforin expressing CD4+ adenovirus-specific T-cells with artificial antigen presenting cells.

Adenoviruses (HAdV) can cause life threatening infections, especially in paediatric patients after allogeneic stem cell transplantation (SCT). Yet, no effective antiviral medication is available. One treatment option is adoptive transfer of HAdV-specific T-cells from the graft donor into the patient. Especially CD4+ T-cells are critical to control HAdV infection. To allow for applicability of CD4+ T-cells in adoptive therapy, sufficient numbers of HAdV-specific T-cells with low levels of residual alloreactive T-cells are required. In this study, we explored the possibility to selectively expand and isolate functional HAdV-specific T-cells from PBMCs in response to 15-mer peptides using artificial antigen-presenting cells (aAPCs), composed of liposomes harbouring HAdV-peptide/HLA-Class-II complexes. HAdV-specific T-cells generated using this method produce mainly pro-inflammatory cytokines, express perforin and granzyme B, kill HAdV-infected cells effectively and are not alloreactive. Thus, the generation and isolation of HAdV-specific CD4+ T-cells seem a critical step towards specific adoptive therapy for HAdV infections after allogeneic SCT.

FOXP3+ CD4+ Tregs lose suppressive potential but remain anergic during transient inflammation in human.

Tregs are crucial in controlling inflammation. Although the transcription factor FOXP3 is the most applicable phenotype marker of Tregs, it does not indisputably characterize suppressive function during T-cell activation in vitro. A question that remains is: what is the functionality of FOXP3(+) T cells during inflammation in vivo? We studied FOXP3(+) T cells in a human model of acute inflammation due to cardiac surgery. Twenty-five children who underwent cardiac surgery for correction of a septum defect were included. Following surgery, we observed a transient systemic inflammatory response accompanied by an increased proportion of CD25(bright) T cells with sustained Treg phenotype. During this transient immune activation, both the percentage of CD4(+) FOXP3(+) cells and the level of expression of FOXP3 in the CD4(+) CD25(bright) CD127(low) population increased. While Tregs remained present during systemic inflammation and continued to be anergic, the capacity to suppress effector T cells was reduced. The reduced suppressive state of Tregs could be induced in vitro by plasma obtained during the peak of inflammation after surgery. These data show that inflammation inhibits Treg function through soluble factors present in plasma. These results underscore the functional role of FOXP3(+) Tregs during inflammation in vivo.

TLR9 agonist CpG enhances protective nasal HSP60 peptide vaccine efficacy in experimental autoimmune arthritis.

OBJECTIVES: Peptide-based immune tolerance induction is considered an attractive treatment option for autoimmune diseases. The authors have developed a novel method that can enhance the induction of protective peptide-specific T-cell responses, using a rat arthritis model. The authors focused on the Toll-like receptor 9 ligand CpG, which was shown to stimulate regulatory T-cell proliferation when added to plasmacytoid dendritic cells (pDC) using in-vitro cultures. METHODS: The peptide used is a heat shock protein 60 epitope (p1) that elicits tolerogenic peptide-specific immune responses in human arthritis patients and was recently shown to have protective capacity as a bystander antigen in the rat adjuvant arthritis model. Rats were treated with three nasal doses of p1, CpG or a combination of p1 and CpG. Antigen-presenting cells were studied in nose-draining lymph nodes (mandibular lymph nodes; MLN) after nasal treatment, and T-cell responses were analysed in joint-draining lymph nodes after arthritis induction. RESULTS: Nasal co-administration of p1/CpG significantly augmented the arthritis-protective effect of p1, while CpG treatment alone did not. Co-treatment of p1/CpG increased both the number and activation status of pDC in draining MLN, which was accompanied by amplified p1-specific T-cell proliferation and interleukin (IL)-10 production. During early arthritis, p1-specific IL-10 production was identified at the site of inflammation. P1 and p1/CpG-treated rats showed a greater amount of CD4+FoxP3+ regulatory T cells in the joint-draining lymph nodes, which correlated with lower arthritis scores. CONCLUSIONS: These clinical and immunological data suggest the use of CpG as a potent adjuvant for mucosal peptide-specific immune therapy in arthritis.

High prevalence of methotrexate intolerance in juvenile idiopathic arthritis: development and validation of a methotrexate intolerance severity score.

OBJECTIVE: To design and validate a new questionnaire for identifying patients with methotrexate (MTX) intolerance, and to determine the prevalence of MTX intolerance in patients with juvenile idiopathic arthritis (JIA) using this questionnaire. METHODS: The MTX Intolerance Severity Score (MISS) questionnaire was constructed, consisting of 5 domains: stomach ache, nausea, vomiting, sore mouth, and behavioral symptoms. The domains each consisted of 3 questions pertaining to the presence of a symptom upon, prior to (anticipatory), and when thinking of (associative) MTX intake. The MISS questionnaire was validated in 86 patients by determining its discriminative power between patients with and those without MTX intolerance, identified as such by a gold standard (physician's opinion). Using the MISS questionnaire, the prevalence of MTX intolerance was determined in 297 JIA patients. RESULTS: The MISS questionnaire discriminated well between MTX-intolerant and MTX-tolerant patients. A cutoff score of 6 yielded the best sensitivity (88%) and specificity (80%). MTX intolerance was found in 150 (50.5%) of 297 patients. Of 220 patients receiving oral MTX, 98 (44.5%) experienced MTX intolerance, whereas 67.5% of 77 patients receiving parenteral MTX experienced intolerance to the drug (P = 0.001). CONCLUSION: Our findings indicate that the MISS questionnaire is a highly sensitive and specific tool for the diagnosis of MTX intolerance, and that there is a high prevalence of MTX intolerance among JIA patients. The prevalence of intolerance in patients receiving parenteral MTX exceeds that in patients receiving oral MTX. The frequent occurrence of anticipatory and associative symptoms suggests that classic conditioning plays an important role in MTX intolerance.

CD30 discriminates heat shock protein 60-induced FOXP3+ CD4+ T cells with a regulatory phenotype.

In many animal models, the manifestations of inflammatory diseases can be prevented by the adoptive transfer of CD4(+)FOXP3(+) regulatory T cells (Tregs). CD4(+)FOXP3(+) Tregs can be obtained by isolation and expansion of polyclonal naturally occurring Tregs or by Ag-specific activation of CD4(+)CD25(-)FOXP3(-) T cells. Two major obstacles are hampering the translation of this latter protocol into therapeutic application. First, there is a lack of knowledge on relevant autoantigens. Second, the resulting population is contaminated with activated CD4(+) T cells that transiently express Forkhead box P3 but gain no regulatory function. Therefore, these cells may not be safe for clinical application. In this study, we demonstrate that highly suppressive FOXP3(+) Tregs can be induced in vitro by the activation of CD4(+)CD25(-) T cells with the self-Ag human 60-kDa heat shock protein (HSP60). The activation induced suppressive FOXP3(+) Tregs can be distinguished by surface expression of CD30 from nonsuppressive FOXP3(+) effector cells. We confirm that the induced CD30(+)FOXP3(+) Tregs recognize HSP60 epitopes and that the induction of Tregs by HSP60 is enhanced by signaling via TLR4 on APCs. These findings have implications for the generation and isolation of pure populations of Ag-specific Tregs, with the potential to prevent and treat human inflammatory diseases.

Vaccination leads to an aberrant FOXP3 T-cell response in non-remitting juvenile idiopathic arthritis.

OBJECTIVE: To investigate how meningococcal C vaccination in patients with remitting (oligoarticular) or progressive (polyarticular) juvenile idiopathic arthritis (JIA) influences the specific T-cell response to both the vaccine and heat shock protein 60, a regulatory auto-antigen in JIA. METHODS: Twenty six oligoarticular, 28 polyarticular JIA patients and 20 healthy adults were studied before and after MenC vaccination in a prospective follow-up study. T-cell proliferation assay, flow cytometry, carboxyfluorescein diacetate succinimidyl ester staining and multiplex immunoassay were performed to quantify and qualify the antigen-specific immune responses. RESULTS: Peripheral blood mononuclear cells (PBMC) from polyarticular JIA exemplified higher antigen-specific CD4 T-cell proliferation, interleukin 2 (IL-2) and tumour necrosis factor alpha (TNFα) production when compared with oligoarticular JIA or healthy individuals after vaccination. Furthermore, in polyarticular JIA antigen-induced CD4+CD25(bright) or CD4+FOXP3+ T cells did not increase upon vaccination. CONCLUSION: Polyarticular JIA CD4+FOXP3+ T cells did not respond to vaccination and demonstrated a higher percentage of cells irrespective of vaccination when compared with oligoarticular JIA. These cells are either activated T cells and/or regulatory cells unable to regulate the antigen-specific immune response after vaccination. When compared with oligoarticular JIA, the increased IL-2 and TNFα production underline the immune hyperresponsiveness of polyarticular JIA PBMC to an antigenic trigger. As this may hold a risk for derailment, these findings could provide a cellular basis for the presumed relationship between environmental triggers and disease in human autoimmune diseases.

Bystander suppression of experimental arthritis by nasal administration of a heat shock protein peptide.

OBJECTIVES: Mucosal immune therapy with disease-inducing antigens is an effective way to prevent experimental arthritis, but in humans these antigens are unknown. In juvenile idiopathic arthritis, however, T cell recognition of a so-called bystander antigen, heat shock protein 60 (HSP60), is associated with a good prognosis. Recently epitopes derived from HSP60, a microbial peptide (p1) and its self-homologue (p2) were reported to induce tolerogenic T cell responses in vitro in patients with arthritis. A study was undertaken to determine whether mucosal administration of these bystander epitopes can be similarly effective in suppressing arthritis. METHODS: Rats were treated nasally with p1, p2 or phosphate-buffered saline before arthritis induction. Arthritis scores were assessed and peptide-specific proliferative responses, phenotypic analysis, cytokine production and in vitro suppressive capacity of cells were measured in lymph nodes and spleens. CD4 spleen T cells from p1- or p2-treated rats were adoptively transferred into naïve rats that were subsequently injected with complete Freund's adjuvant for arthritis induction. RESULTS: Nasal administration of p1 prevented experimental arthritis whereas treatment with the self-homologue p2 did not. Adoptive transfer of CD4 T cells protected against experimental arthritis. Treatment with p1 increased peptide-specific and self-crossreactive interferon γ (IFNγ) production. Tumour necrosis factor α (TNFα) levels were reduced at the site of inflammation. Forkhead box P3 (FoxP3) expression remained stable but the suppressive capacity of T regulatory cells in p1-treated rats was enhanced. CONCLUSION: p1 immune therapy induces a population of CD4 T cells with reduced TNFα and increased peptide-specific IFNγ production at the site of inflammation. This population expresses FoxP3 and has potent suppressive capacity which, upon transfer, protects against arthritis. The bystander epitope p1 may therefore be a suitable candidate for antigen-specific immunotherapy in arthritis.

Mutations in the perforin gene can be linked to macrophage activation syndrome in patients with systemic onset juvenile idiopathic arthritis.

OBJECTIVE: Macrophage activation syndrome (MAS) in systemic onset juvenile idiopathic arthritis (SoJIA) is considered to be an acquired form of familial haemophagocytic lymphohistiocytosis (fHLH). FHLH is an autosomal recessive disorder, characterized by diminished NK cell function and caused by mutations in the perforin gene (PRF1) in 20-50% of patients. Interestingly, SoJIA patients display decreased levels of perforin in NK cells and diminished NK cell function as well. Here, we analysed PRF1 and its putative promoter in SoJIA patients with or without a history of MAS. METHODS: DNA of 56 SoJIA patients (41 Italian and 15 Dutch) was isolated. Of these, 15 (27%) had a confirmed history of MAS. We sequenced PRF1 and 1.5 kb of the 5'-upstream region. DNA sequence variations in the promoter region were functionally tested in transfection experiments using a human NK cell line. RESULTS: We detected a previously undescribed sequence variation (-499 C > T) in the promoter of PRF1 in 18% of the SoJIA patients. However, transfection experiments did not show functional implications of this variation. Secondly, we found that 11 of 56 (20%) SoJIA patients were heterozygous for missense mutations in PRF1. In particular, we found a high prevalence of the Ala91Val mutation, a variant known to result in defective function of perforin. Interestingly, the prevalence of Ala91Val in SoJIA patients with a history of MAS (20%) was increased compared with SoJIA patients without MAS (9.8%). One SoJIA patient, heterozygous for Ala91Val, showed profound decreased perforin levels at the time of MAS. CONCLUSIONS: These findings suggest that PRF1 mutations play a role in the development of MAS in SoJIA patients.

The effect of menaquinone-7 (vitamin K2) supplementation on osteocalcin carboxylation in healthy prepubertal children.

Vitamin K contributes to bone health, probably through its role as cofactor in the carboxylation of osteocalcin. Intervention studies in adults have demonstrated that markedly higher osteocalcin carboxylation is obtained by intakes of vitamin K well above the current recommended dietary intake. However, the relationship between increased vitamin K2 intake and enhanced osteocalcin carboxylation has never been shown in healthy children. The objective was to study the effect of 45 microg menaquinone-7 (MK-7; one of the vitamin K2 species) on the circulating levels of undercarboxylated osteocalcin (ucOC) and carboxylated osteocalcin (cOC) in healthy prepubertal children. We hypothesised that MK-7 supplementation will reduce the ucOC:cOC ratio (UCR), indicating an improved vitamin K status. The present study is a double-blind randomised placebo-controlled trial examining the effect of 8 weeks MK-7 supplementation on the carboxylation of osteocalcin in healthy children (n 55). Serum levels of ucOC, cOC and MK-7 were measured at baseline and after 8 weeks, together with bone markers and coagulation parameters. The UCR was used as an indicator of vitamin K status. In the MK-7-supplemented group (n 28), the circulating concentration of inactive ucOC reduced and the UCR improved whereas the concentration of MK-7 increased. Within the placebo group, ucOC, cOC, UCR and MK-7 did not significantly change over time. In both groups, bone markers and coagulation parameters remained constant over time. These findings demonstrate that in healthy, prepubertal children, modest supplementation with MK-7 increases circulating concentrations of MK-7 and increases osteocalcin carboxylation.

Vitamin K status is associated with childhood bone mineral content.

In adult bone, vitamin K contributes to bone health, probably through its role as co-factor in the carboxylation of osteocalcin. In children, the significance of vitamin K in bone-mass acquisition is less well known. The objective of this longitudinal study was to determine whether biochemical indicators of vitamin K status are related to (gains in) bone mineral content (BMC) and markers of bone metabolism in peripubertal children. In 307 healthy children (mean age 11.2 years), BMC of the total body, lumbar spine and femoral neck was determined at baseline and 2 years later. Vitamin K status (ratio of undercarboxylated (ucOC) to carboxylated (cOC) fractions of osteocalcin; UCR) was also measured at both time points. Markers of bone metabolism, sex steroids, vitamin D status and growth hormones were measured at baseline only. Large variations in the levels of the UCR were found at both time-points, indicating a substantial interindividual difference in vitamin K status. Improvement of vitamin K status over 2 years (n 281 children) was associated with a marked increase in total body BMC (r -49.1, P<0.001). The UCR was associated with pubertal stage, markers of bone metabolism, sex hormones and vitamin D status. A better vitamin K status was associated with more pronounced increase in bone mass in healthy peripubertal children. In order to determine the significance of these findings for childhood bone health, additional paediatric studies are needed.

Self-investigation to explore the impact of juvenile arthritis on adolescent life: a case-study.

OBJECTIVE: To gain insight into the personal experience and feelings of an adolescent with a chronic disease. METHODS: We report on the application of the self-confrontation method (SCM), illustrated by a case-example of an adolescent with juvenile idiopathic arthritis. RESULTS: Although taken at face value she was not impeded by the arthritis, through self-assessment with the SCM this adolescent acknowledged and addressed the emotional struggle to keep the arthritis secret and to constantly test the physical limits of her body. After the process of self-reflection, the adolescent showed a better integration of her arthritis experiences into her life story. CONCLUSION: With the SCM the adolescent could explore her own functioning and well-being on a manifest, as well as on an emotional and motivational level. PRACTICE IMPLICATIONS: In future research, by studying the self-investigations of a group of adolescents with chronic diseases, common risk factors for the development of a stable identity during adolescence might be identified. In clinical care, the SCM promotes self-knowledge, allowing for an intrinsic motivation to deal with the emotional impact of the disease.

Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.

BACKGROUND: X-linked severe combined immunodeficiency due to a mutation in the gene encoding the common gamma (gamma(c)) chain is a lethal condition that can be cured by allogeneic stem-cell transplantation. We investigated whether infusion of autologous hematopoietic stem cells that had been transduced in vitro with the gamma(c) gene can restore the immune system in patients with severe combined immunodeficiency. METHODS: CD34+ bone marrow cells from five boys with X-linked severe combined immunodeficiency were transduced ex vivo with the use of a defective retroviral vector. Integration and expression of the gamma(c) transgene and development of lymphocyte subgroups and their functions were sequentially analyzed over a period of up to 2.5 years after gene transfer. RESULTS: No adverse effects resulted from the procedure. Transduced T cells and natural killer cells appeared in the blood of four of the five patients within four months. The numbers and phenotypes of T cells, the repertoire of T-cell receptors, and the in vitro proliferative responses of T cells to several antigens after immunization were nearly normal up to two years after treatment. Thymopoiesis was documented by the presence of naive T cells and T-cell antigen-receptor episomes and the development of a normal-sized thymus gland. The frequency of transduced B cells was low, but serum immunoglobulin levels and antibody production after immunization were sufficient to avoid the need for intravenous immunoglobulin. Correction of the immunodeficiency eradicated established infections and allowed patients to have a normal life. CONCLUSIONS: Ex vivo gene therapy with gamma(c) can safely correct the immune deficiency of patients with X-linked severe combined immunodeficiency.

Personality and chronic fatigue syndrome: methodological and conceptual issues.

Among clinical psychologists, consulting physicians, scientific researchers and society in general an image has emerged of patients with chronic fatigue syndrome (CFS) as perfectionist, conscientious, hardworking, somewhat neurotic and introverted individuals with high personal standards, a great desire to be socially accepted and with a history of continuously pushing themselves past their limits. The aim of this article is to (a) give a concise review of the main recent studies on personality and CFS, (b) address the major methodological problems in the study of personality in CFS and (c) discuss some of the conceptual assumptions that seem to limit the research on personality and CFS. The results of the reviewed studies range from no evidence of major differences between the personalities of patients with CFS and controls, to evidence of severe psychopathology and personality disorder in patients with CFS. Although personality seems to play a role in CFS, it is difficult to draw general conclusions on the relation between personality and CFS. It is argued that this is partially due to the diversity and heterogeneity in study methods, patient populations, control groups and CFS case definitions. Personality should be regarded as an important factor to be studied in CFS. However, additional studies are needed, not focusing exclusively on personality disorder, or personality considered on a general trait level. In recent developments in personality research, the continually evolving life narrative that makes sense of, and gives direction to, an individual's life is also regarded as an important aspect of personality. New insights into personality and CFS might be gained by systematically studying the self-narratives of patients with the syndrome.

Alexithymia in adolescents with chronic fatigue syndrome.

BACKGROUND: Alexithymia is postulated as an important factor in the development of medically unexplained physical symptoms. Chronic fatigue syndrome (CFS) is presently medically unexplained. The aim of this study was to investigate whether the prevalence of alexithymia was higher in adolescents with CFS compared to healthy adolescents. Comorbidity such as anxiety and depression were analyzed as possible confounding factors. Secondly, alexithymia was investigated as a prognostic factor for the recovery of CFS. METHODS: A cross-sectional study was performed among 40 adolescent outpatients diagnosed with CFS and 36 healthy controls. The 20-item Toronto Alexithymia Scale was used to assess all participants for alexithymia. Additionally, all participants completed a number of questionnaires regarding fatigue (Checklist Individual Strength), somatic complaints (Checklist Somatization Inventory), depression (Children's Depression Inventory), and trait anxiety (Spielberger State Trait Anxiety Questionnaire). A follow-up study was performed among the CFS adolescents 1 1/2 years after the initial assessment. RESULTS: CFS adolescents scored higher only on the subscale identifying feelings of the TAS-20 [mean difference after adjustment for depression and anxiety 2.8 (95% CI: 0.6; 4.9]. Twelve CFS adolescents (30%) fulfilled criteria for alexithymia. This subgroup was characterized by higher scores for depression and anxiety and equal scores for fatigue and somatic complaints. At follow-up, no differences in recovery were established between the alexithymic and nonalexithymic CFS adolescents. CONCLUSIONS: Alexithymia neither appears to be a unique correlate of CFS nor to be a prognostic factor for recovery of the CFS illness.

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.

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.

Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.

Cytokines, chemokines and soluble adhesion molecules interact in a complex network within the immune system. Fingerprinting of these proteins may allow the use of these proteins as biomarkers for identification of disease, disease subtyping and monitoring therapeutic interventions. We developed a multiplex immunoassay (MIA) for the detection of 30 proteins in a variety of human body fluids such as plasma and synovial fluid (SF). The measurement of these proteins is hampered by the presence of human (auto-) antibodies, which can cause non-specific binding. We have validated a novel approach for the removal of interfering immunoglobulins using pre-absorption with protein-L. Interfering (auto-) antibodies, such as rheumatoid factor (RF), were removed using three methods; polyethylene glycol (PEG) precipitation, pre-absorption with human gamma-globulin or pre-absorption with protein-L. A significant decrease of RF was observed after a 2 h incubation with protein-L. RF IgM levels were reduced by 89% whereas total IgM, IgG and IgA levels were reduced by 60%. Residual immunoglobulins were blocked with rodent serum and did not interfere with the multiplex immunoassay. Comparing the MIA with a conventional enzyme-linked immunosorbent assay (ELISA) using a panel of spiked plasma samples resulted in correlation coefficients for all mediators between R2 = 0.88 and R2 = 0.99. Intra-assay variance was less than 10% whereas inter-assay variance ranged between 6% and 16%. Pathological samples with heterophilic antibodies hamper immunoassays such as ELISA and MIA. We show that pre-absorption with protein-L is a powerful tool for removal of interfering immunoglobulins from human bodily fluids to be used in immunoassays for studying changes in protein patterns.

Nasopharyngeal pneumococcal carriage after combined pneumococcal conjugate and polysaccharide vaccination in children with a history of recurrent acute otitis media.

BACKGROUND: We recently showed that vaccination with a 7-valent pneumococcal conjugate vaccine (PCV7) followed by a 23-valent pneumococcal polysaccharide vaccine (PPSV23) failed to prevent new episodes of acute otitis media (AOM) in previously unvaccinated toddlers and children with a history of recurrent AOM. We describe in detail the impact of pneumococcal vaccinations on nasopharyngeal carriage of S. pneumoniae in this study population. METHODS: The impact of vaccination with PCV7 followed by PPSV23 on pneumococcal nasopharyngeal carriage was studied in a prospective, randomized trial involving 383 children (age range, 1-7 years) with previous AOM. Nasopharyngeal swab specimens were collected at the time of first vaccination and at 6-7-month intervals during the 26-month follow-up period. RESULTS: Overall, pneumococcal carriage rates did not diminish, remaining at approximately 50% in both PCV7/PPSV23 and control vaccinees. A significant shift from conjugate vaccine- to nonconjugate vaccine-type pneumococci was observed in children aged 1-2 years, who received the conjugate vaccine twice before the polysaccharide vaccine was administered. Conjugate vaccine serotype carriage was not influenced in older children, who received the conjugate vaccine once before receiving the polysaccharide booster. CONCLUSIONS: The administration of conjugate vaccines at least twice also after 2 years of age may be mandatory for reducing the carriage of conjugate vaccine serotypes in children with recurrent AOM. Polysaccharide booster vaccination did not affect nasopharyngeal colonization with serotypes not included in the conjugate vaccine.

General and segmental reduced pain thresholds in juvenile chronic arthritis.

The objective of this work was to investigate the pattern of pain threshold (PT) alterations in paraspinal soft tissues as related to inflamed joints in patients with juvenile chronic arthritis (JCA). A pressure algometer was used in JCA patients with inflamed knee(s) (n = 16), with inflamed ankle(s) (n = 17), and in healthy controls (n = 69), all aged 6-17 years. Local pressure was applied to the joint capsules of the knees and ankles and the soft paraspinal tissues, and PTs were recorded. JCA patients showed not only a significantly lower PT at the inflamed knee and ankle joints, but also in the non-inflamed paraspinal areas. All paraspinal PTs showed a significant negative correlation with the inflamed knee or ankle joint. The correlation rank order showed that the paraspinal L1 area had the highest negative correlation with the inflamed knee joint and the paraspinal L3 area with the inflamed ankle joint. JCA coincides with generally diminished pain thresholds in the paraspinal region. Nociception from inflamed joints may have established changes in the peripheral as well as central nociceptive processing system in JCA.

Stem cell transplantation for autoimmune disorders. Refractory juvenile idiopathic arthritis.

Since 1997, haematopoietic stem cell transplantation (HSCT) has been applied as an experimental procedure in more than 50 children with refractory juvenile idiopathic arthritis. We describe here follow-up data on 34 children with juvenile idiopathic arthritis, treated with HSCT in order to evaluate its feasibility, safety and efficacy. Data were collected on immunological reconstitution, complications and key rheumatological parameters. The clinical follow-up of the children ranged from 12 to 48 months. Eighteen of the 34 patients achieved a drug-free complete remission. Seven of these patients had previously failed treatment with anti-tumour necrosis factor-alpha. Six of the 34 patients showed a partial response (ranging from 30 to 70%), and 7 of the 34 patients showed a complete relapse of disease. Infectious complications were frequently seen. There were three cases of transplant-related mortality and two cases of disease-related mortality. It is still unclear why especially patients with systemic juvenile idiopathic arthritis are at risk of episodes of reactive haemophagocytosis.