Phenotypic and functional characterization of in vitro cultured human mast cells.

BACKGROUND: Mast cell progenitor cells, derived from CD34+ hematopoietic stem cells, enter the circulation and subsequently mucosal or connective tissues where they mature to mast cells. Upon activation, mast cells increase the expression of activation markers, e.g. CD63, and release histamine amongst other mediators. Traditionally, release of these mediators is quantified using assays measuring their extracellular concentration in the supernatant of stimulated cells. METHODS: Human mast cells (HuMC) were cultured from peripheral blood, phenotypically characterized, passively sensitized with allogenic IgE antibodies and finally stimulated by anti-IgE that crosslinks IgE/FcεRI complexes. Alterations in the number of cells positive for CD63 and release of histamine were quantified simultaneously by flow cytometry. RESULTS: In culture, two distinct CD45+ cell populations were identified: CD117+ CD203c+hi and CD117- CD203c+low cells. Both populations showed positivity for FcεRI, tryptase and chymase, and contained histamine. Activation resulted in a significant increase of cells positive for CD63+ up to 21% (range: 11-39) for CD117+ CD203c+hi cells (P = 0.005), and 27% (18-55) CD63+ for CD117- CD203c+low cells (P = 0.02). Baseline histamine content was higher for CD117+ CD203c+hi cells than for CD117- CD203c+low cells, respectively 994 (695-6815) Molecules of Equivalent Specific Fluorochrome V500 per cell (MESF-V500/cell) and 797 (629-4978) MESF-V500/cell (P = 0.02). After activation, CD117+ CD203c+hi cells showed significant histamine release of 578 (366-1521) MESF-V500/cell, whilst CD117- CD203c+low cells resulted in 310 (217-366) MESF-V500/cell histamine release. CONCLUSION: This study discloses that culturing HuMC from CD34+ progenitors yields 2 phenotypically distinct cell populations that display a greatly similar response upon cross-linking of IgE/FcεRI complexes. © 2016 International Clinical Cytometry Society.

Cannabis sativa allergy: looking through the fog.

IgE-mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life-threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross-allergies, mostly for plant foods. This clinical entity, designated as the 'cannabis-fruit/vegetable syndrome', might also imply cross-reactivity with tobacco, natural latex and plant-food-derived alcoholic beverages. Hitherto, these cross-allergies are predominantly reported in Europe and appear mainly to rely upon cross-reactivity between nonspecific lipid transfer proteins or thaumatin-like proteins present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross-allergies remains uncertain.

Flowcytometric diagnosis of atracurium-induced anaphylaxis.

BACKGROUND: Allergy to atracurium is a rare condition with serious consequences of diagnostic error. However, correct diagnosis is not always straightforward. The aim of this study is to assess the utility of the basophil activation test (BAT) in atracurium sensitization and to investigate its role in identifying cross-reactivity between muscle relaxants. METHODS: For validation, eight patients with perioperative anaphylaxis to atracurium and seven individuals experiencing perioperative anaphylaxis but not exposed to neuromuscular blocking agents (NMBA) were included. Furthermore, five other patient groups were included in the study, and all individuals exposed to different NMBA, either sensitized or not to the drug. Basophil activation with atracurium was analysed flow cytometrically. RESULTS: ROC analyses between eight atracurium-sensitized patients and seven nonexposed controls allowed identification of 5% as the decision threshold for BAT positivity. For this cutoff, the BAT attained a sensitivity of 63%, specificity of 100%, positive predictive value of 100% and negative predictive value of 70%. Of the atracurium-exposed individuals with a negative atracurium skin test (ST), two individuals had a clear positive BAT. BAT atracurium was positive in one cisatracurium-sensitized patient and negative in all cisatracurium-exposed patients with a negative ST to cisatracurium. All rocuronium- and suxamethonium-sensitized patients displayed a negative BAT with atracurium. CONCLUSIONS: The BAT proves to be a useful diagnostic for atracurium-induced anaphylaxis and may be complementary to STs. The technique enables quick and simultaneous testing of potentially crossreactive NMBA and the identification of safe alternatives for future surgery.

Basophilic histamine content and release during venom immunotherapy: insights by flow cytometry.

BACKGROUND: Despite the efficiency of venom immunotherapy, the effects on basophils and mast cells remain incompletely understood and probably vary according to the treatment phase. OBJECTIVES: To study the effect of build-up and maintenance venom immunotherapy on individual basophils. METHODS: Intracellular histamine and its release was analyzed flow cytometrically by a new enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells included flow cytometric quantification of CD63 and CD203c. Analyses of basophil activation experiments were performed before the start of treatment, after build-up therapy and during maintenance therapy. RESULTS: Before the start of therapy, patients demonstrated significantly higher numbers of basophils when compared with stung control individuals. At the end of build-up therapy a decrease of basophil numbers was observed, whereas during maintenance therapy basophil counts returned to pretreatment values. Before the start of therapy, the intracellular histamine content per cell in patients was significantly higher when compared with stung control individuals. During maintenance therapy intracellular histamine content decreased to values observed in stung control individuals. In addition, maintenance therapy lowered the net release of histamine per cell in response to optimal stimulation with wasp venom. CONCLUSIONS: We introduce a novel technique that enables to assess the effects of venom immunotherapy on basophils. This new technique may help to monitor treatment effects in individual patients and could aid in the development of more efficient and better tolerated immunotherapy protocols.

IgE-mediated allergy to pholcodine and cross-reactivity to neuromuscular blocking agents: Lessons from flow cytometry.

BACKGROUND: Immunoglubulin E antibody-mediated allergic reactions to opioids are rare and difficult to document correctly. OBJECTIVE: Assessment of the basophil activation test in the diagnosis of IgE-mediated allergy to the antitussive pholcodine and associated sensitizations to neuromuscular blocking agents (NMBA). METHODS: Three patients with a suspected IgE-mediated allergy to pholcodine were investigated using skin tests, quantification of specific IgE, and flow cytometric activation of basophils. RESULTS AND CONCLUSION: Flow cytometric activation of basophils, with simultaneous analysis of CD63 appearance and median histamine content per cell, is the only technique capable to correctly document pholcodine allergy. The negative predictive value of basophil activation tests might help to elucidate on the controversial putative cross-reactivity between pholcodine and NMBA.

Multicentric plasma cell variant Castleman's disease presenting with cutaneous vasculitis and pulmonary parenchymal involvement in a patient with ankylosing spondylitis: case report and review of the literature.

We present a case of the multicentric plasma cell variant of Castleman's disease (CD) with two rare manifestations. The patient consulted us because of cutaneous vasculitis of the lower limbs, while constitutional symptoms were nearly absent. Imaging studies also revealed pulmonary parenchymal involvement. Furthermore, our patient is the first case in whom association of ankylosing spondylitis with CD is reported. In addition, we present a review of the literature with emphasis on the clinical presentation of CD and its difficult discrimination from autoimmune and infectious disorders. An overview of the therapeutic options is also provided.

Human basophils: a unique biological instrument to detect the allergenicity of food.

BACKGROUND: Labeling of major food allergens is mandatory for the safety of allergic consumers. Although enzyme-linked immunosorbent assay, polymerase chain reaction, and mass spectrometry are sensitive and specific instruments to detect trace amounts of food proteins, they cannot measure the ability of food constituents to trigger activation of mast cells or basophils. AIM: We evaluated the basophil activation test as an instrument to determine the allergenic potential of trace amounts of food allergens in complex matrices. Peanut (Arachis hypogaea) allergy was selected as a proof-of-concept model. METHODS: The study population comprised 5 severely peanut-allergic patients (3 males/2 females; median age, 12 years) all sensitized to 3 major peanut allergens (Ara h 1, Ara h 2, and Ara h 3) and 5 peanut-tolerant individuals (2 males/3 females; median age, 8 years). Basophils from patients and controls were stimulated with pure peanut extract and blank and peanut-spiked (0.1, 0.01, and 0.001 ppm) biscuits (baking time 11, 16, 21, 26 minutes) and chocolate extracts. RESULTS: Blank biscuits and chocolate did not induce cell activation in patients or controls. A comparison between patients and controls showed significantly higher activation of basophils after stimulation with 0.1 and 0.01 ppm of peanut-spiked biscuit at all baking times and peanut-spiked chocolate (P < .05). CONCLUSIONS: The basophil activation test is a highly sensitive and specific tool to detect traces of functionally active food allergens. For biscuits, its accuracy seems independent of baking time. Furthermore, it allows even the most sensitive patients to be included in study protocols.

Multicentric reticulohistiocytosis associated arthritis responding to anti-TNF and methotrexate.

We present a patient with therapy resistant multicentric reticulohistiocytosis (MRH). MRH is a rare granulomatous, multisystem disease characterised most frequently by disfiguring papulonodular skin lesions and sometimes a destructive polyarthritis, though any organ can be involved. Abnormal histiocytic reactions to an undetermined stimulus (possibly an associated mycobacterial infection, auto immune process or neoplastic process) have been proposed as an underlying mechanism. The diagnosis is confirmed by histopathology of the cutaneous nodules and/or synovial membrane by the presence of CD68-positive histiocytes and multinucleated giant cells with an eosinophilic 'ground-glass' cytoplasm. Recent studies have identified TNFalpha and other inflammatory cytokines to be highly expressed in the synovium and synovial fluid of affected joints in patients with MRH. Based on these findings, we treated our patient with infliximab in combination with methotrexate with marked improvement of morning stiffness, tender and swollen joint count, visual analogue scale and health assessment questionnaire after his third infusion. However, the nodules did not markedly resolve. When treating patients with MRH with TNFa neutralizing drugs, one has to keep the possible association with malignancy in 15-30% of cases in mind and these products should be used with caution.

Vanishing tumour of the colon ascendens due to acquired type II C1-inhibitor deficiency.

We present a patient with recurrent bouts of angioedema of the lips, throat and extremities with a negative familial history for angioedema. Laboratory results confirmed an angioedema due to acquired C1-INH deficiency (or acquired angioedema, AAE). As AAE can result from underlying disease, further investigation toward malignancy was initiated. A CT-scan of the abdomen disclosed a circumferential tumour of the proximal segment of the colon ascendens which disappeared by the time an ileocolonoscopy was executed. Angioedema of the bowel has been widely reported in hereditary angioedema, whereas it is anecdotal in AAE.

T cell signal transducer and activator of transcription (STAT) 4 and 6 are affected by adalimumab therapy in rheumatoid arthritis.

OBJECTIVES: TNF-alpha inhibition therapy affects the systemic immune response in rheumatoid arthritis by influencing T cell subtypes (Th1, Th2, Treg), but its effect on the intracellular signal transduction in T cells remains largely unexplored. Here we studied the activation of Th1-associated signalling molecule STAT4 and Th2-associated STAT6 in CD4+ T cells. METHODS: Eight rheumatoid arthritis patients were studied before and after 12 weeks of adalimumab therapy and compared to 8 healthy individuals. Peripheral blood mononuclear cells (PBMC) were analysed flow cytometrically either directly after isolation or after 24 hours of anti-CD3/anti-CD28 stimulation, to determine spontaneous and IL-4/IL-12-induced STAT4 and STAT6 phosphorylation in CD4+ T cells. Cytokine production by stimulated PBMC was measured in the supernatant using a cytometric bead array. Non-parametric statistical tests were applied. RESULTS: After adalimumab therapy, phospho-STAT6 increased, both in freshly isolated and anti-CD3/anti-CD28-stimulated CD4+ T cells. The STAT6 response to brief IL-4 stimulation did not change. In healthy individuals and adalimumab-treated patients, anti-CD3/anti-CD28 induced the phosphorylation of STAT4, but not in untreated patients. IFN-gamma production in untreated patients was significantly lower than in healthy individuals or adalimumab-treated patients. In contrast, the production of IL-4, IL-6 and IL-12 was not influenced. CONCLUSIONS: Adalimumab therapy increases Th2-associated STAT6 phosphorylation and restores the activation-induced STAT4 phosphorylation to the levels in healthy individuals. This advocates against a pro-inflammatory effect of Th1-associated STAT4 and might provide an explanation for the influence of TNF inhibition therapy on the systemic T cell response in rheumatoid arthritis.

Angioedema beyond histamine: an educational case series.

Angioedema constitutes an important clinical problem that can cause significant morbidity and mortality. Correct management requires a prompt recognition and treatment of the acute event and identification of the underlying cause. Many cases are caused by non-allergic reactions and do not result from mediator release by degranulating mast cells and basophils, but are related to accumulation of plasma and tissue bradykinin. This case series aims primarily to describe some important causes of non-allergic bradykinin-induced angioedema. Particular emphasis is put on clinical particularities, differential diagnosis, diagnostic approach and correct therapeutic management, as bradykinin-mediated angioedema is unresponsive to antihistamines.

Performance of automated measurement of antibodies to cyclic citrullinated peptide in the routine clinical laboratory.

OBJECTIVE: To evaluate the performing technical and clinical characteristics of an automated system for routine measurement of anticyclic citrullinated peptide antibodies (aCCP), a new marker for rheumatoid arthritis (RA). MATERIAL AND METHODS: Reproducibility, repeatability and linearity of aCCP, as measured by an automated fluorescent enzyme immunoassay (FEIA/Phadia), were evaluated and compared with the performance of a manual ELISA technique (Axis Shield Diagnostics). Clinical verification of both methods included estimation of sensitivity in RA patients (n = 42) and specificity in well-characterized non-RA autoimmune disease controls (n = 49) and healthy subjects (n = 39). RESULTS: Precision studies showed a coefficient of variation between 4.9 % and 10 % for the FEIA technique and between 6.35% and 19% for the ELISA technique. Both systems showed good linear response. Sensitivity of aCCP for RA was 74% for FEIA and 79% for ELISA. Specificity was 100% for both methods, as calculated for healthy subjects. For non-RA-diseased controls, specificities of 98% and 94% were obtained for FEIA and ELISA, respectively. Both methods were concordant in 97% of cases. Increasing the cut-off for the ELISA system from >5 U/mL to >11 U/mL resulted in lower sensitivity (71.4%) but higher specificity (98.0%), i.e. improved discriminating power between RA and non-RA and 100% agreement between both methods. CONCLUSION: Automated FEIA measurement of aCCP in the routine clinical laboratory improves imprecision compared to the manual ELISA. Our preliminary results suggest that an increase in cut-off for the ELISA can improve specificity to RA from 94% to 98 %.

Combined analysis of intracellular signalling and immunophenotype of human peripheral blood basophils by flow cytometry: a proof of concept.

BACKGROUND: The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood. OBJECTIVES: To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry. METHODS: Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed. RESULTS: Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation. CONCLUSIONS: This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. CAPSULE SUMMARY: This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.

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.

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.

Intestinal necrosis in a patient with rheumatoid arthritis receiving anti-TNF treatment.

Vasculitis leading to intestinal necrosis is a rare complication of rheumatoid arthritis. The introduction of anti-TNF treatment for methotrexate-resistant cases improved disease-control substantially in these often more aggresive forms of rheumatoid arthritis. As far as we know only two cases of severe vasculitis following anti-TNF treatment have been reported. We describe a 45-year old female patient with severe rheumatoid arthritis, who presented with an epileptic insult, renal failure and a quickly deteriorating general condition due to intestinal vasculitis, while she had been receiving anti-TNF treatment for 6 months.

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.

Perinatal risk factors for sensitization, atopic dermatitis and wheezing during the first year of life (PIPO study).

OBJECTIVE: To evaluate the influence of perinatal environmental factors on early sensitization, atopic dermatitis and wheezing during the first year. METHODS: Information on pregnancy-related factors, parental atopic history, environmental factors and the clinical course of the infant until age one was gathered by questionnaires, as part of a prospective birth cohort study (Prospective study on the Influence of Perinatal factors on the Occurrence of asthma and allergies [PIPO-study]). Quantification of total and specific IgE was performed in 810 children and their parents. RESULTS: Early sensitization was found in 107/810 (13%) of the infants. Multiple regression analysis showed that specific IgE in fathers was a risk factor for early sensitization in their daughters (adjusted odds ratios (OR(adj)) 2.21 (95% confidence interval (CI) 1.10-4.49); P=0.03), whereas in boys, day care attendance was shown to be protective for early sensitization (OR(adj) 0.38 (95% CI 0.20-0.71); P=0.001). Atopic dermatitis occurred in 195/792 infants (25%). Specific IgE in the mother (OR(adj) 1.52 (95% CI 1.06-2.19); P=0.02) and in the infant (OR(adj) 4.20 (95% CI 2.63-6.68); P<0.001) were both risk factors for the occurence of atopic dermatitis, whereas postnatal exposure to cats was negatively associated with atopic dermatitis (OR(adj) 0.68 (0.47-0.97); P=0.03). Postnatal exposure to cigarette smoke (OR(adj) 3.31 (95% CI 1.79-6.09); P<0.001) and day care attendance (OR(adj) 1.96 (95% CI 1.18-3.23); P=0.009) were significantly associated with early wheezing, which occurred in 25% (197/795) of the infants. CONCLUSION: The effect of paternal sensitization and day care attendance on sensitization is gender dependent. Maternal sensitization predisposes for atopic dermatitis, whereas postnatal exposure to cats had a protective effect.