Translation, cross-cultural adaptation, and validation of the Mouth Handicap in Systemic Sclerosis questionnaire (MHISS) into the Dutch language.

The Mouth Handicap in Systemic Sclerosis (MHISS) is a French-generic questionnaire evaluating mouth-opening restriction, dryness, and esthetic concerns. The aim of this study was to translate and adapt the MHISS questionnaire into the Dutch language and evaluate its psychometric properties. The MHISS was translated according to international guidelines, field-tested among 16 systemic sclerosis (SSc) patients, and adapted. Subsequently, the Dutch MHISS was administered to 52 SSc patients visiting the outpatient or day patient clinic of a university hospital and readministered after 2 weeks. Internal consistency was tested by computing Cronbach's alpha. Test-retest reliability was determined by computing the intraclass correlation coefficient (ICC) and validity by determining associations with measures of overall functioning (Health Assessment Questionnaire (HAQ)), maximum mouth opening (MMO, in millimeter), subjective xerostomia (visual analog scale), and objective xerostomia (Saxon test). Patients had mean ± standard deviation (SD) age and disease duration of 55 ± 21 and 7.2 ± 7.3 years. Twenty-seven (52 %) patients had diffuse cutaneous SSc. The mean Dutch MHISS score was 17.5 (SD 10.0) with Cronbach's alpha being 0.862. Dutch MHISS scores differed significantly between patients with high and low disability levels (HAQ, MMO, and subjective and objective xerostomia divided according to the median; paired t test). Spearman rank correlations with HAQ (r = 0.599, p = 0.000), MMO (r = -0.518, p = 0.000), and subjective xerostomia (r = 0.536, p = 0.000) were moderate; correlation with objective xerostomia did not reach statistical significance. The ICC was 0.94. The Dutch version of the MHISS demonstrated good psychometric properties and is useful in assessing mouth disability in SSc patients.

The autoimmune disease-associated IL2RA locus is involved in the clinical manifestations of systemic sclerosis.

Regulatory T cells (T(regs)) are crucial in the maintenance of the immune tolerance and seem to have an important role in systemic sclerosis (SSc). The interleukin 2 receptor α (IL2RA) is an important T(reg) marker, and polymorphisms of IL2RA gene are associated with a number of autoimmune diseases. Therefore, we aimed to investigate for the first time the association of the IL2RA locus in SSc. For this purpose, a total of 3023 SSc patients and 2735 matched healthy controls, from six European Caucasian cohorts, were genotyped for the IL2RA gene variants rs11594656, rs2104286 and rs12722495 using the TaqMan allelic discrimination technology. The overall meta-analysis reached statistical significance when the three polymorphisms were tested for association with SSc, the limited subtype (lcSSc) and anti-centromere auto-antibodies (ACAs). However, no significant P-values were obtained when the ACA-positive patients were removed from the SSc and lcSSc groups, suggesting that these associations rely on ACA positivity. The strongest association signal with ACA production was detected for rs2104286 (P(FDR)=2.07 × 10(-4), odds ratio=1.30 (1.14-1.47)). The associations of rs11594656 and rs12722495 were lost after conditioning to rs2104286, and allelic combination tests did not evidence a combined effect, indicating that rs2104286 best described the association between IL2RA and ACA presence in SSc.

Anti-citrullinated protein antibodies in rheumatoid arthritis: a functional role for mast cells and basophils?

Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation of the joints. Anti-citrullinated protein antibodies (ACPA) are highly specific for RA and are associated with a more severe disease progression. ACPA have also been shown to have a pathological role in RA. In animal models of RA, ACPA enhances arthritis. Furthermore, in vitro generated immune complexes with ACPA can activate macrophages and the complement system in the human system. Recently, a direct functional and specific response of FcεRI+ immune cells towards citrullinated proteins was demonstrated. Basophils of ACPA+ RA patients are activated by citrullinated proteins that cross link the FcεRI receptor via IgE-ACPA, physiologically bound to the receptor. In addition, synovial mast cells from ACPA+ RA patients show a more activated phenotype than mast cells from ACPA- patients. These findings underline the suggestion that ACPA+ and ACPA- RA are two different disease entities and point to a possible functional role of ACPA and FcεRI+ cells in the pathogenesis of RA.

Randomized comparison of a multidisciplinary team care program with usual care in patients with systemic sclerosis.

OBJECTIVE: To compare the effectiveness of a multidisciplinary team care program with usual outpatient care in patients with systemic sclerosis (SSc; scleroderma). METHODS: We performed a randomized controlled trial comparing a 12-week multidisciplinary team care program (1 day per week; individual treatments, group exercises, and group education) with outpatient clinic care. Outcome measures included the Hand Mobility in Scleroderma (HAMIS) test, grip strength, maximal mouth opening (MMO), 6-minute walk distance (6MWD), maximum aerobic capacity (VO(2max) ), Checklist Individual Strength 20 (CIS-20), SSc Health Assessment Questionnaire (HAQ), and Short Form 36 (SF-36), assessed at 0, 12, and 24 weeks. Statistical comparisons of change scores were done by analysis of covariance. RESULTS: Twenty-eight patients were assigned to the intervention group (mean age 53.9 years, 15 of 28 with diffuse SSc) and 25 were assigned to the control group (mean age 51.7 years, 15 of 25 with diffuse SSc). Twenty-five patients (89%) in the intervention group completed the treatment program. At 12 weeks, there was a significantly greater improvement in grip strength (2.2 versus -1.8 kg; P = 0.001), MMO (1.4 versus -0.9 mm; P = 0.011), 6MWD (42.8 versus 3.9 meters; P = 0.021), and HAQ score (-0.18 versus 0.13; P = 0.025) in the intervention group, whereas differences for the other outcome measures did not reach significance. At 24 weeks, the effect on grip strength persisted. CONCLUSION: In patients with SSc, a 12-week multidisciplinary day patient treatment program was more effective than regular outpatient care with respect to 6MWD, grip strength, MMO, and HAQ score, but not for VO(2max) , HAMIS test, CIS-20, SF-36, and visual analog scale for pain. This study provides a first step in quantifying the effect of a multidisciplinary team care program and warrants the conduct of further intervention studies.

Evidence for a functional role of IgE anticitrullinated protein antibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease involving inflammation of the joints. Among the autoantibodies described in RA, anticitrullinated protein antibodies (ACPAs) are highly specific and predictive for RA. In addition, ACPAs have been implicated in the pathogenesis of RA. However, a direct functional response of immune cells from ACPA(+) RA patients toward citrullinated proteins has not been demonstrated. In this study, we show that exposure to citrullinated antigens leads to activation of basophils from ACPA(+) RA patients within 20 minutes. This was not observed after exposure of basophils to noncitrullinated control antigens or after stimulation of basophils from ACPA(-) RA patients and healthy controls. Basophil activation was correlated with the binding of citrullinated proteins to basophils. Furthermore, serum from ACPA(+) RA patients in contrast to that from ACPA(-) RA patients could specifically sensitize human FcepsilonRI expressing rat basophil cells (RBL), enabling activation by citrullinated proteins. Mast cell degranulation products such as histamine levels were enhanced in synovial fluid of ACPA(+) RA patients as compared with ACPA(-) RA and osteoarthritis patients. In addition, histamine levels in synovial fluid from ACPA(+) RA patients correlated with IgE levels, suggesting degranulation of mast cells by cross-linking IgE. Immunohistochemistry on synovial biopsies demonstrated an increased number of degranulated CD117(+) mast cells in ACPA(+) RA patients; IgE and FcepsilonRI expression in synovial mast cells from ACPA(+) RA patients was increased. In conclusion, our results show an immunological response of immune cells from ACPA(+) RA patients in a citrulline-specific manner. Moreover, these data indicate a role for IgE-ACPAs and FcepsilonRI-positive cells in the pathogenesis of RA.

Influence of simvastatin on the production of pro-inflammatory cytokines and nitric oxide by activated human chondrocytes.

OBJECTIVE: In addition to their cholesterol-lowering action, statins have been suggested to exert anti-inflammatory activities. In this study we evaluate whether simvastatin could influence the production of pro-inflammatory cytokines (interleukin (IL)-6 and IL-8) and nitric oxide (NO) by activated human chondrocytes. METHODS: Human isolated chondrocytes and cartilage explants were pre-incubated with simvastatin (0.5, 5, 10 and 50 micromol/L) for 48 h. Then the cultures were stimulated with a mixture of IL-1Beta and TNF-alpha (10 ng/mL) and co-incubated with simvastatin for an additional 48 h. A flow cytometric microsphere-based immunoassay was performed to detect cytokine secretion in the supernatants. NO production was quantified using the Griess assay. RESULTS: Simvastatin demonstrated significant dose-dependent inhibition of IL-6 and IL-8 production of isolated chondrocytes and cartilage explants up to 99% for IL-6 and up to 88% for IL-8 (p < 0.01). At the higher concentrations simvastatin decreased NO production by both isolated chondrocytes (up to 43%, p < 0.01) and cartilage explants (up to 30%, p < 0.01). CONCLUSION: This study demonstrates anti-inflammatory properties of simvastatin in chondrocytes in vitro, suggesting a potential cartilage-protective role for statins in arthritis.

Synovial fluid and peripheral blood immune complexes of patients with rheumatoid arthritis induce apoptosis in cytokine-activated chondrocytes.

The destruction of cartilage is an important characteristic of rheumatoid arthritis (RA). Immune complexes (IC) are usually found in high amounts in RA synovial fluids (SF) and in the superficial layers of RA cartilage. The objective of this study was to investigate if IC have a direct influence on proliferation, survival and production of nitric oxide (NO) of cytokine-activated chondrocytes. Primary bovine chondrocytes were incubated with cytokines (huIL-1alpha, bovIFN-gamma, huTNF-alpha) and IC containing precipitates of peripheral blood (PB) and/or synovial fluid (SF) of 14 RA patients, 5 osteoarthritis (OA) patients and 10 healthy age and sex-matched controls. After 48 h, chondrocyte viability, proliferation, apoptosis, NO production and oxygen radical levels were measured. Staining with May-Grünwald-Giemsa after incubation with IC of RA PB and SF, showed apoptotic chondrocytes with condensation of the nuclei. The proliferation rates of cytokine-activated chondrocytes, incubated with sera and SF IC of RA patients were significantly decreased compared to chondrocytes, incubated with sera and SF IC of OA patients and compared to sera of controls. Quantitative evaluation of apoptotic cells by annexin-V/propidium iodide and TUNEL assays revealed a significant increase after incubation with sera and SF IC of RA patients, compared to control sera and OAs sera and SF. In all TUNEL positive samples, active-caspase-3-positive cells were found. There was a significant increase of chondrocyte NO production, after incubation with SF IC of RA patients, compared to OA SF. These results support the hypothesis that IC, present in serum and SF of RA patients, have a profound influence on chondrocyte growth, NO production and apoptosis, contributing to cartilage destruction in RA.

Wegener's granulomatosis presenting as a thyroid mass.

A 71-year-old patient was referred for suspected hyperthyroidism because of a 15-kg weight loss, suppressed thyroid stimulating hormone (TSH), and a 4-cm nodule in the left thyroid lobe. Both free T4 and T3 were normal. Antithyroglobulin, anti-TSH receptor and antimicrosomal antibodies were absent. Thyroid scintigraphy showed a cold nodule in the left thyroid lobe. CAT scan of the neck revealed a 4-cm inhomogeneous nodule at the left side. An elevated sedimentation rate suggested bacterial thyroiditis, localized Quervain thyroiditis, malignancy, and the fibrosing variant of Hashimoto's thyroiditis or Riedel's thyroiditis. A fine needle biopsy of the thyroid nodule showed no malignant cells but was inconclusive. A true cut biopsy demonstrated atypical inflammation and also failed to reveal the diagnosis. Therefore, the patient was admitted to the hospital for further work-up and was unexpectedly found to have nodular lesions in the lung on a chest X-ray. Additional blood analysis revealed a positive cytoplasmic ANCA-titer. After inconclusive peripheral lung biopsies, a left hemithyroidectomy and a very large video-assisted thoracoscopic lung biopsy were performed, both revealing extensive zones of necrosis surrounded by granulomatous foci pointing to the diagnosis of Wegener's granulomatosis (WG) disease. To our knowledge, this is the first report of a well-documented WG of the thyroid gland. Although extremely rare, WG should be included in differential diagnosis of inflammatory lesions of the thyroid gland.

Flow-assisted quantification of in vitro activated basophils in the diagnosis of wasp venom allergy and follow-up of wasp venom immunotherapy.

BACKGROUND: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy (VIT). Despite the efficacy of VIT, issues as how to monitor treatment and when to discontinue maintenance therapy remain to be established. METHODS: To evaluate diagnostic performances of the basophil activation test (BAT) in wasp venom allergy, 80 patients with a definite history of wasp venom anaphylaxis (systemic reactors) and 14 wasp-stung asymptomatic controls (stung controls) were enrolled. Venom-induced basophil activation was analyzed flow cytometrically by double-labeling with anti-IgE and anti-CD63. Results were compared to wasp IgE levels and results of a venom skin test (VST). To establish whether the BAT constitutes a candidate marker to monitor VIT, the BAT was repeated in 22 patients on the 5th day of a build-up course and after 6 months of maintenance VIT. Whether the BAT could contribute in the decision of discontinuing VIT was assessed in a cross-sectional analysis in 30 patients receiving treatment for 3 years. RESULTS: Comparison between systemic reactors and stung controls revealed a sensitivity of 86.4% and specificity of 100% for venom IgE, and sensitivity of 81.8% for VST, respectively. In contrast to stung controls, patients demonstrated dose-dependent venom-induced basophil activation. The BAT attained a sensitivity of 83.8% and specificity of 100%. At the end of the build-up course, no effect of VIT on the BAT was demonstrable. When the BAT was repeated after 6 months of treatment, submaximal stimulation of the cells demonstrated a significant decreased CD63 expression (P < 0.04). Patients having VIT for 3 years also demonstrated significantly lower venom-induced CD63 expression (P < 0.001). After 3 years, 60% of the patients had a negative BAT for submaximal stimulation of the cells whereas only 17.9% of the patients had negativation of wasp IgE. CONCLUSIONS: The BAT is a reliable instrument for the diagnosis of wasp venom anaphylaxis and might constitute an instrument to monitor wasp VIT.

Effect of bisphosphonates on nitric oxide production by inflammatory activated chondrocytes.

OBJECTIVE: Bisphosphonates have been reported to possess anti-inflammatory and cartilage protective effects in animal arthritis models but not much is known about their direct effect on chondrocytes. In this study we evaluate the effect of bisphosphonates on nitric oxide (NO) production by activated chondrocytes. METHODS: Isolated bovine chondrocytes and bovine cartilage explants were used. In the second part of the study human cartilage explants (osteoarthritis (OA) and non-OA cartilage) were used. The isolated chondrocytes and cartilage explants were pre-incubated with clodronate, pamidronate or risedronate and stimulated with IL-1 and TNF-alpha (10 ng/mL, 48 h). NO production was quantified using the Griess assay. RESULTS: In bovine cultures, clodronate (10(-4)mol/L) and pamidronate (10(-6)mol/L) showed a small inhibition of NO production (up to 15 % and 25% respectively), whereas risedronate had no effect. In the human cartilage cultures no effect of BPs on the NO production was detected except for the highest concentration of clodronate tested (10(-4)mol/L) which demonstrated a small enhancement (19%) in NO production reaching significance in the non-OA group. CONCLUSION: BPs have a modest effect on NO production by inflammatory activated chondrocytes only in the higher concentrations, indicating that the clinical relevance of these effects is probably negligible.

Flow-assisted allergy diagnosis: current applications and future perspectives.

Physicians predominantly rely upon quantification of serum-specific immunoglobulin E (IgE) and/or skin test to confirm clinically suspected IgE-mediated allergy. However, for various reasons, identification of the offending allergen(s) and potentially cross-reactive structures is not always straightforward. Flow-assisted allergy diagnosis relies upon quantification of alterations in the expression of particular basophilic activation markers. Actually, upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. Currently, the technique has been applied in the investigation of IgE-mediated allergy caused by classical inhalant allergens, food, Hevea latex, hymenoptera venoms and drugs. It is also appreciated; the technique proves valuable in the diagnosis of non-IgE-mediated (anaphylactoid) reactions such drug hypersensitivity and the detection of autoantibodies in certain forms of chronic urticaria. This review will not address immunologic features, characteristics and general pitfalls of flow-assisted analysis of in vitro-activated basophils as summarized elsewhere. After a recapitulation of the principles and some specific technical issues of flow-assisted analysis of in vitro-activated basophils, we principally focus on the current clinical and research applications of the basophil activation tests. Personal experience of both research groups is provided, where appropriate. Finally, a viewpoint on how the field might evolve in the following years is provided.

Antioxidant effect of bisphosphonates and simvastatin on chondrocyte lipid peroxidation.

The objective of this study was to evaluate the effect of bisphosphonates (BPs) and simvastatin on chondrocyte lipid peroxidation. For this purpose, a flow cytometrical method using C11-BODIPY(581/591) was developed to detect hydroperoxide-induced lipid peroxidation in chondrocytes. Tertiary butylhydroperoxide (t-BHP) induced a time and concentration dependent increase in chondrocyte lipid peroxidation. Addition of a Fe2+/EDTA complex to t-BHP or hydrogen peroxide (H2O2) clearly enhanced lipid peroxidation. The lipophilic simvastatin demonstrated a small inhibition in the chondrocyte lipid peroxidation. None of three tested BPs (clodronate, pamidronate, and risedronate) had an effect on chondrocyte lipid peroxidation induced by t-BHP. However, when Fe2+/EDTA complex was added to t-BHP or H2O2, BPs inhibited the lipid peroxidation process varying from 25% to 58%. This study demonstrates that BPs have antioxidant properties as iron chelators, thereby inhibiting the chondrocyte lipid peroxidation. These findings add evidence to the therapeutic potential of bisphosphonates and statins in rheumatoid arthritis.

Influence of anti-tumor necrosis factor therapy (Adalimumab) on regulatory T cells and dendritic cells in rheumatoid arthritis.

OBJECTIVE: To investigate whether anti-TNF therapy could have an effect on dendritic cells (DCs) and regulatory T cells in rheumatoid arthritis (RA) patients. METHODS: A four-colour flow cytometric technique was used to measure CD4+CD25+ T cells i.e. CD4+CD25high+ (regulatory T cells) and CD4+CD25low+ (activated T cells)), DCs as well as the in vitro, intracellular, lipopolysaccharide-stimulated cytokine production of DCs. RESULTS: Clinical and laboratory parameters of disease activity decreased after anti-TNF treatment. Before anti-TNF therapy, RA patients demonstrated a decreased count of Th2-promoting lymphoid DCs as compared to controls and after anti-TNF therapy this decrease was sustained. Intracellular cytokine production was only found in the myeloid DCs population and there was a higher production of TNF-alpha and IL1-b as compared to healthy controls. Treatment did not alter this cytokine production. Before anti-TNF therapy, the percentage CD4+CD25+ T cells was significantly elevated in RA patients than in healthy controls. CONCLUSION: These results demonstrate anti-TNF to be a potent anti-inflammatory drug, as mirrored by the decrease in clinical and biological parameters as well as the decrease in activated CD4+ T cells. However, in this study no demonstrable effect on DCs and regulatory T cells was found.

In vitro allergy diagnosis: should we follow the flow?

During the last 5 years, an increasing number of studies have demonstrated that flow cytometric quantification of in vitro basophil activation can be a quite performant and reliable tool to measure IgE-dependent allergen-specific responses in allergic patients. So far, most assays have used CD63 as a basophil activation marker and native allergen extracts for stimulation. However, other basophil markers and recombinant allergens have recently been introduced. The technique has been applied for the diagnosis of allergy to pollen, house dust mite, food, natural rubber latex, hymenoptera venom and drugs. In addition, the technique has proven to be useful in non-IgE-mediated reactions such as hypersensitivity to drugs as well as detection of auto-antibodies in chronic urticaria. This review will focus on some specific issues: (1) principles of flow cytometric analysis of in vitro-activated basophils, (2) general technical aspects of the technique (including passive sensitization), (3) clinical applications and (4) recommendations for further development and evaluation of the technique.

Selective in vitro antioxidant properties of bisphosphonates.

The aim of this study was to investigate the in vitro antioxidant profile of different bisphosphonates. Bisphosphonates were tested for their xanthine oxidase and microsomal lipid peroxidation inhibiting capacity. Furthermore, the effect of these different compounds on DPPH, a stable radical, was investigated. Clodronate, risedronate, and pyrophosphate were further tested for their hydroxyl radical scavenging activity. None of the tested compounds showed xanthine oxidase inhibiting activity or DPPH scavenging activity. All the tested bisphosphonates exhibited inhibiting capacities on the microsomal lipid peroxidation. The hydroxyl radical scavenging activity was dependent on the order of adding the different reagents and was highest for risedronate. Bisphosphonates possess an inhibiting activity on the microsomal lipid peroxidation and the Fenton reaction. In these reactions iron plays an important role suggesting that the selective in vitro antioxidant properties of the bisphosphonates are due to their iron chelating characteristics.

Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques.

BACKGROUND: We investigated the relation between intracellular cytokine production and extracellular cytokine levels by using two flow cytometric techniques. METHODS: A two-color flow cytometric technique was used to measure interleukin (IL)-1beta, IL-6, tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-12 production blocked intracellularly with brefeldin A in lipopolysaccharide (LPS)-stimulated CD14(+) monocytes and IL-2, IL-4, and IFN-gamma production in phorbol-12-mirystate-13-acetate (PMA)-stimulated CD3(+) T lymphocytes in samples from patients with rheumatoid arthritis. A flow cytometric microsphere-based immunoassay was performed to detect cytokine secretion in plasma of PMA- and LPS-stimulated whole blood samples. RESULTS: There was a strong linear correlation between extracellular quantitative (pg/ml) and intracellular semiquantitative detection of LPS-stimulated IL-1beta, IL-6, IL-10, and IL-12 production (r > 0.9). For lymphocytes, extracellularly detected IL-2 and IFN-gamma correlated well with percentages of cytokine-producing cells (r > 0.8). The percentages of IL-4-positive T cells were moderately correlated with the secreted amounts of IL-4 as detected with the microsphere-based immunoassay (r = 0.7). CONCLUSION: Overall, there was a good correlation between semiquantitative intracellular detection of cytokines and the secreted amounts of cytokines detected with the microsphere based immunoassay.

Influence of pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammatory (IL-4) cytokines on chondrocyte function.

OBJECTIVE: Cytokines produced by inflammatory cells play a pivotal role in synovial inflammation and joint destruction in rheumatoid arthritis. To investigate the influence of pro-inflammatory cytokines (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and subsequently the possible beneficial role of an anti-inflammatory cytokine (IL-4) on chondrocyte viability (necrosis/apoptosis), proliferation and nitric oxide (NO) production. METHODS: Primary bovine chondrocytes were cultured until monolayers were obtained. Cells were incubated with cytokines (IL-1 alpha, IFN-gamma, TNF-alpha, IL-4, IL-6) at 0.1, 1, 10 and 100 ng/mL. After 48 h, the viability of the chondrocytes was measured flow cytometrically with propidium iodide. Proliferation was determined by the incorporation of tritiated thymidine. The morphology of the chondrocytes, including presence of apoptotic nuclei, was evaluated by a May-Grünwald-Giemsa staining. In addition, the number of apoptotic chondrocytes was detected flow cytometrically with a TUNEL technique and annexin-V/propidium iodide staining. NO production was evaluated using a spectrophotometric assay, based upon the Griess reaction. RESULTS: The viability and proliferation of bovine chondrocytes decreased after incubation with 100 ng/mL IL-1 alpha, TNF-alpha or IFN-gamma. In contrast, incubation of chondrocytes with IL-4 or IL-6 had no influence on the viability or the proliferation of cells. IL-1 alpha was able to enhance NO production in a dose dependent manner. IFN-gamma and TNF-alpha induced NO production only at the highest concentration (100 ng/mL), whereas IL-4 and IL-6 did not. There was a dose dependent increase in apoptosis of bovine chondrocytes cultured in the presence of IL-1 alpha and TNF-alpha. This effect could not be prevented by preincubation with IL-4. Preincubation with IL-4 diminished IL-1 alpha and TNF-alpha induced NO production and increased proliferation of chondrocytes. In an additional experiment, incubation of human chondrocytes with anti-Fas did not induce apoptosis as measured by annexin-V/propidium iodide staining. CONCLUSIONS: Pro-inflammatory cytokines are able to induce apoptosis, whereas IL-4 as an anti-inflammatory cytokine can inhibit the effect of IL-1 alpha and TNF-alpha on NO production and proliferation of bovine chondrocytes.

Differences in circulating dendritic cell subtypes in cord blood and peripheral blood of healthy and allergic children.

BACKGROUND: Different types of circulating dendritic cells have been described. Dendritic cells influence differentiation of naive T lymphocytes into T helper type 1 (Th1) and Th2 effector cells. OBJECTIVE: The purpose of this study was to evaluate the number of circulating DC subtypes in peripheral blood of allergic and healthy children and in cord blood of neonates from allergic and non-allergic parents. METHODS: Circulating dendritic cells were flow cytometrically identified in whole blood samples as lineage (CD3, CD14, CD16, CD19, CD20, CD56) negative, CD34 negative and HLA-DR-positive cells. According to the expression of CD123 and CD11c, different DC subtypes were identified. RESULTS: Apart from DC1 (CD11c+ CD123dim+) and DC2 (CD11c- CD123high+), a third DC population was described with less differentiated phenotypic characteristics, namely CD11c- CD123dim+, and therefore defined here as less differentiated DC (ldDC). These ldDC represented the major DC population in cord blood and showed an age-depended decrease. The highest level of ldDC was detected in children with atopic dermatitis, whereas asthmatic children showed the lowest ldDC counts. Furthermore, high-dose inhaled corticosteroid treatment in asthmatic children was related to a decreased ldDC number. The number of circulating DC2 was significantly lower in allergic children, especially in asthmatics, compared to healthy children. In cord blood, no differences in DC subtypes were detectable between neonates at low and high risk for allergic disorders. CONCLUSION: These results indicate that, apart from DC1 and DC2, a third population of dendritic cells, identified as CD11c- CD123dim+ cells and defined as less differentiated DC, must be considered in the evaluation of circulating DC. Furthermore, DC2 counts were decreased in allergic children, especially in asthmatics, which might be the consequence of an increased recruitment to the target organs.