To cite this article: Schaub A, von Gunten S, Vogel M, Wymann S, Rüegsegger M, Stadler BM, Spycher M, Simon H‐U, Miescher S. Dimeric IVIG contains natural anti‐Siglec‐9 autoantibodies and their ...anti‐idiotypes. Allergy 2011; 66: 1030–1037.
Background: Intravenous immunoglobulin (IVIG) preparations are increasingly used for the treatment of autoimmune and chronic inflammatory diseases. Naturally occurring autoantibodies against Siglec‐9 and Fas are thought to contribute to the anti‐inflammatory effects of IVIG via cell death regulation of leukocytes and tissue cells. Dimeric IVIG fractions are suspected to contain idiotypic (Id)‐anti‐idiotypic complexes of antibodies, which might also include anti‐Siglec‐9 and anti‐Fas autoantibodies.
Methods: Dimeric IVIG fractions were separated from monomeric IVIG by size‐exclusion chromatography and remonomerized by low pH treatment. Binding studies of total, monomeric, and dimeric IVIG were performed using surface plasmon resonance and flow cytometry on primary human neutrophils.
Results: Anti‐Siglec‐9 and anti‐Fas autoantibodies were contained in both monomeric and dimeric IVIG fractions, but anti‐Siglec‐9 antibodies were highly enriched in dimeric IVIG. The propensity to engage in dimer formation was paratope dependent. IVIG binding to Siglec‐9 was specific and sialylation independent. Interestingly, we detected anti‐idiotypic antibodies (anti‐Ids) against anti‐Siglec‐9 autoantibodies in dimeric, but not in monomeric fractions of IVIG.
Conclusions: Our study supports the concept that idiotype–anti‐idiotype (Id–anti‐Id) interactions contribute to the dimer formation in IVIG preparations. To our knowledge, this is the first description of Id–anti‐Id dimers of death receptor‐specific antibodies in IVIG. Such Id–anti‐Id interactions might determine the activity of immunomodulatory antibodies present both in IVIG and the patient.
Background
Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their ...low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought‐after tool for diagnostic and drug testing purposes.
Methods
A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin‐3 (IL‐3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression.
Results
IL‐3‐dependent, conditional Hoxb8‐immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near‐unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end‐differentiated and short‐lived and express basophil‐specific surface markers and proteases. Upon IgE‐ as well as C5a‐mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2‐type cytokines (IL‐4, IL‐13) and leukotriene C4. IL‐3‐deprivation induces apoptosis correlating with upregulation of the BH3‐only proteins BCL‐2‐interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM‐1).
Conclusion
A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.
Background: Interferon (IFN)‐α is a cytokine that possesses potent anti‐viral and immunoregulatory activities. We aimed to assess clinical and immunological effects of low‐dose IFN‐α in patients ...with severe corticosteroid‐resistant asthma with and without Churg–Strauss syndrome. There is currently no efficient pharmacological treatment available for this group of patients.
Methods: We studied 10 patients with corticosteroid‐resistant asthma, in which 3×106 IU/day IFN‐α were administrated in addition to the prednisone dose given already before introduction of the cytokine therapy. The prednisone dose was gradually reduced dependent on the clinical situation and used as a clinical readout to evaluate the efficacy of the cytokine therapy. To distinguish between IFN‐α‐ and prednisone‐mediated immunological changes, the corticosteroid dose was kept constant for at least 2 weeks upon introduction of the cytokine therapy in seven patients. The effects of treatment on clinical and immunological parameters were measured at 2–4 weeks and 5–10 months depending on the availability of the patient.
Results: Interferon‐α treatment rapidly improved the clinical situation as assessed by lung function parameters and required prednisone dose. Important immunological changes included: decreased leukocyte numbers, increased relative numbers of CD4+ T cells, increased differentiation of T helper (Th)1 cells, and increased expression of interleukin (IL)‐10 in peripheral blood mononuclear cells.
Conclusion: Interferon‐α treatment was associated with dramatic improvements in the condition of patients with corticosteroid‐resistant asthma with and without Churg–Strauss syndrome. Potential mechanisms of action include the establishment of a correct Th1/Th2 balance and the induction of the anti‐inflammatory IL‐10 gene.
G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, ...but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.
Background
The aim of this Delphi study was to reach consensus about definition, operationalization and assessment of visual pursuit (VP) and visual fixation (VF).
Methods
In a three-round ...international Delphi study, clinical and research experts on disorders of consciousness indicated their level of agreement on 87 statements using a 5-point Likert scale. Consensus for agreement was defined by a median of 5, an interquartile range (IQR) ≤ 1, and ≥ 80% indicating moderate or strong agreement.
Results
Forty-three experts from three continents participated, 32 completed all three rounds. For VP, the consensus statements with the highest levels of agreement were on the term ‘pursuit of a visual stimulus’, the description ‘ability to follow visually in horizontal and/or vertical plane’, a duration > 2 s, tracking in horizontal and vertical planes, and a frequency of more than 2 times per assessment. For VF, consensus statements with the highest levels of agreement were on the term ‘sustained VF’, the description ‘sustained fixation in response to a salient stimulus’, a duration of > 2 s and a frequency of 2 or more times per assessment. The assessment factors with the highest levels of agreement were personalized stimuli, the use of eye tracking technology, a patient dependent time of assessment, sufficient environmental light, upright posture, and the necessity to exclude ocular/oculomotor problems.
Conclusion
This first international Delphi study on VP and VF in patients with disorders of consciousness provides provisional operational definitions and an overview of the most relevant assessment factors.
The cause of persistent eosinophilia and the hypereosinophilic syndrome is unknown. Recent work suggests that in some patients with the hypereosinophilic syndrome, a clone of abnormal T cells ...produces large amounts of interleukin-5, a cytokine required for the growth and differentiation of eosinophils. We examined T-cell surface markers, rearranged T-cell-receptor genes, and in vitro production of cytokines by T cells from patients with idiopathic eosinophilia.
The expression of surface molecules on T cells was measured by flow cytometry. Cytokine expression was measured by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analysis. To identify dominant (clonal) rearrangements of the T-cell receptor within the lymphocyte population, Southern blot analysis (beta chain) and the polymerase chain reaction (gamma chain) were performed according to standard protocols.
Among 60 patients with idiopathic eosinophilia, 16 had circulating T cells with an aberrant immunophenotype. In each of these patients, the abnormal immunophenotype was unique. Evidence of clonal rearrangements of the T-cell receptor was obtained in 8 of the 16 patients. In most instances, the abnormal T cells expressed large amounts of surface proteins associated with T-cell activation (the alpha chain of the interleukin-2 receptor and the HLA-DR antigen). Moreover, the aberrant T cells produced large amounts of interleukin-5 in vitro.
Clonal populations of abnormal T cells producing interleukin-5 occur in some patients with idiopathic eosinophilia.
Zusammenfassung
Die eosinophilen Granulozyten sind als Untergruppe der Leukozyten ein Teil des angeborenen Immunzellpools. Zusätzlich nehmen sie homöostatische Aufgaben im Gewebe war. ...Klassischerweise werden Allergien und Parasiteninfektionen mit einer erhöhten Eosinophilenzahl assoziiert, doch findet man eine Eosinophilie auch bei Vaskulitiden und Tumorerkrankungen. Die wichtigsten Steuerungselemente der Eosinophilen sind das Zytokin Interleukin 5 und die Eotaxine. Selbst produzieren Eosinophile die unterschiedlichsten Kommunikationsfaktoren und toxischen Proteine, die in den zytoplasmatischen Granula gespeichert sind und bei Bedarf abhängig vom jeweiligen Stimulus gezielt und schnell ausgeschüttet werden können. Zur Pathogenbekämpfung können Eosinophile auch extrazelluläre mitochondriale DNA-Netze herauskatapultieren. In dieser Übersicht werden Grundaufbau, Steuerung und Funktion der Eosinophilen im Gesunden und bei Krankheiten besprochen.
Idiopathic eosinophilic esophagitis (IEE) is a chronic-inflammatory disorder of the esophagus of unknown origin. The established cornerstone of diagnosis is a dense infiltration of the esophagus with ...eosinophils, but neither the precise pattern of inflammatory cell infiltration nor the mechanisms that likely contribute to induction and maintenance of the inflammatory response have been described.
The intention of this study was to characterize the esophageal inflammatory infiltrate and the expression of cytokines in the esophagus in this disease. In addition, we searched for immunologic abnormalities of blood leukocytes to exclude major primary hyporeactive and hyperreactive conditions of the immune system.
Infiltration of inflammatory cells in the esophagus, stomach, and duodenum was analyzed by immunohistochemistry through use of mAbs against lineage-associated molecules. Cytokine expression was measured by ELISA and immunohistochemical analysis. Lymphocyte subpopulations in blood were determined by means of flow cytometry.
High eosinophil infiltration into the esophageal squamous epithelium was observed in patients with IEE but not in control subjects. Interestingly, increased T-cell and mast cell numbers were also found within the epithelium in these patients. In contrast, the numbers of inflammatory cells were not increased in the stomach and duodenum in patients with IEE, suggesting a specific inflammatory process within the esophagus. Moreover, increased expression of IL-5 and TNF-alpha was observed in esophageal epithelial biopsy specimens. The distribution of lymphocyte subsets in the peripheral blood and their capacity to generate cytokines did not reflect the changes observed at the inflammatory site.
IEE is a selective inflammatory response of the esophagus. T cells, IL-5, eosinophils, and IgE-mediated mechanisms appear to be involved, giving rise to the possibility that allergic reactions might play a role in the pathogenesis of the disease.
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